Biphenyl compounds useful as muscarinic receptor antagonists

ABSTRACT

The invention provides compounds of formula I: 
                         
wherein a, b, c, p, W, A, X 1 , R 1 , R 2 , R 3 , R 6 , R 7 , and R 8  are as defined in the specification. The compounds of formula I are muscarinic receptor antagonists. The invention also provides pharmaceutical compositions containing such compounds, processes and intermediates for preparing such compounds and methods of using such compounds to treat pulmonary disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/660,379, filed on Mar. 10, 2005; the entire disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel biphenyl compounds havingmuscarinic receptor antagonist or anticholinergic activity. Theinvention also relates to pharmaceutical compositions comprising suchbiphenyl compounds, processes and intermediates for preparing suchbiphenyl compounds and methods of using such biphenyl compounds to treatpulmonary disorders.

2. State of the Art

Pulmonary or respiratory disorders, such as chronic obstructivepulmonary disease (COPD) and asthma, afflict many millions of peopleworldwide and such disorders are a leading cause of morbidity andmortality.

Muscarinic receptor antagonists are known to provide bronchoprotectiveeffects and therefore, such compounds are useful for treatingrespiratory disorders such as COPD and asthma. When used to treat suchdisorders, muscarinic receptor antagonists are typically administered byinhalation. However, even when administered by inhalation, a significantamount of the muscarinic receptor antagonist is often absorbed into thesystemic circulation resulting in systemic side effects such as drymouth, mydriasis and cardiovascular side effects.

Additionally, many inhaled muscarinic receptor antagonists have arelatively short duration of action requiring that they be administeredseveral times per day. Such a multiple-daily dosing regime is not onlyinconvenient but also creates a significant risk of inadequate treatmentdue to patient non-compliance with the required frequent dosingschedule.

Accordingly, a need exists for new muscarinic receptor antagonists. Inparticular, a need exists for new muscarinic receptor antagonists thathaving high potency and reduced systemic side effects when administeredby inhalation. Additionally, a need exists for inhaled muscarinicreceptor antagonists having a long duration of action thereby allowingfor once-daily or even once-weekly dosing. Such compounds are expectedto be particularly effective for treating pulmonary disorders, such asCOPD and asthma, while reducing or eliminating side effects such asdry-mouth and constipation.

SUMMARY OF THE INVENTION

The present invention provides novel biphenyl compounds havingmuscarinic receptor antagonist or anticholinergic activity. Among otherproperties, compounds of the invention are expected to possess highpotency and reduced systemic side effects when administered byinhalation and are also expected to possess a long duration of action.

One aspect of the invention relates to a compound of formula I:

where:

a is 0 or an integer of from 1 to 5;

each R¹ is independently selected from (1-4C)alkyl, (2-4C)alkenyl,(2-4C)alkynyl, (3-6C)cycloalkyl, cyano, halo, —OR^(1a), —C(O)OR^(1b),—SR^(1c), —S(O)R^(1d), —S(O)₂R^(1e), —NR^(1f)R^(1g),—NR^(1h)S(O)₂R^(1i), and —NR^(1j)C(O)R^(1k); where each of R^(1a),R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R^(1g), R^(1h), R^(1i), R^(1j),and R^(1k) is independently hydrogen, (1-4C)alkyl or phenyl(1-4C)alkyl;

b is 0 or an integer of from 1 to 4;

each R² is independently selected from (1-4C)alkyl, (2-4C)alkenyl,(2-4C)alkynyl, (3-6C)cycloalkyl, cyano, halo, —OR^(2a), —C(O)OR^(2b),—SR^(2c), —S(O)R^(2d), —S(O)₂R^(2e), —NR^(2f)R^(2g),—NR^(2h)S(O)₂R^(2i), and —NR^(2j)C(O)R^(2k); where each of R^(2a),R^(2b), R^(2c), R^(2d), R^(2e), R^(2f), R^(2g), R^(2h), R^(2i), R^(2j),and R^(2k) is independently hydrogen, (1-4C)alkyl or phenyl(1-4C)alkyl;

W represents O or NW^(a), where W^(a) is hydrogen or (1-4C)alkyl;

c is 0 or an integer from 1 to 5;

each R independently represents (1-4C)alkyl or two R³ groups are joinedto form (1-3C)alkylene, (2-3C)alkenylene or oxiran-2,3-diyl;

A is selected from:

where m is 0 or 1; r is 2, 3 or 4; s is 0, 1 or 2; t is 0, 1 or 2; R⁴ isselected from hydrogen, (1-4C)alkyl, and (3-4C)cycloalkyl; and Ar¹represents a phenylene group or a (3-5C)heteroarylene group containing 1or 2 heteroatoms independently selected from oxygen, nitrogen andsulfur; wherein the phenylene or heteroarylene group is substituted with(R⁵)_(q) where q is 0 or an integer from 1 to 4 and each R⁵ isindependently selected from halo, hydroxy, (1-4C)alkyl and (1-4C)alkoxy;

R⁶ is selected from hydrogen, (1-4C)alkyl, (3-4C)cycloalkyl,—C(O)(1-4C)alkyl, -(1-4C)alkyleneC(O)R^(6a), —C(O)heterocyclyl,—C(O)CH(NH₂)(1-4C)alkyleneQ, -(1-4C)alkyleneC(O)Z, —C(O)(1-4C)alkyleneZ,and —S(O)₂(1-4C)alkyleneZ; where Q is a nitrogen-containing substituentselected from —NR^(6b)R^(6c) and heteroaryl; Z is a nitrogen-containingsubstituent selected from —NR^(6d)R^(6e) and heterocyclyl; R^(6a) ishydrogen or (1-4C)alkyl; each of R^(6b), R^(6c), R^(6d) and R^(6e)independently represents hydrogen, (1-4C)alkyl, (3-6C)cycloalkyl orhydroxyphenyl, and where (1-4C)alkyl is unsubstituted or substituted by1 or 2 substituents independently selected from amido, cyano, furyl,hydroxyl, and methylimidazolyl; the heterocyclyl contains 1 or 2nitrogen atoms, and is unsubstituted or substituted by 1 or 2substituents independently selected from hydroxyl, amido, (1-4C)alkoxy,oxo, —S(O)₂(1-4C)alkyl, —(CH₂)O(1-4C)alkyl, -(1-4C)alkyleneOH,—NR^(6f)R^(6g) and —C(O)NR^(6h)R^(6i), where each of R^(6f), R^(6g),R^(6h) and R^(6i) independently represents hydrogen or (1-4C)alkyl; andthe heteroaryl contains 1 or 2 nitrogen atoms;

X¹ is selected from (1-3C)alkylene, —C(O)(1-3C)alkylene,(1-3C)alkyleneC(O)—, —SO₂—, —SO₂(1-3C)alkylene and (1-3C)alkyleneSO₂—;where the alkylene group in any X¹ is optionally substituted with 1 or 2substituents independently selected from (1-4C)alkyl and —NR^(Xa)R^(Xb);wherein R^(Xa) and R^(Xb) are independently selected from hydrogen and(1-4alkyl);

p is 0, 1 or 2;

each R⁷ independently represents (1-4C)alkyl, (2-4C)alkenyl,(2-4C)alkynyl, (3-6C)cycloalkyl, cyano, nitro, halo,N,N-di(1-4C)alkylamino(2-4C)alkoxy, —OR^(7a), —C(O)OR^(7b), —SR^(7c),—S(O)R^(7d), —S(O)₂R^(7e) or —NR^(7f)R^(7g); R^(7a) is (1-4C)alkyl,(3-6C)cycloalkyl, phenyl or phenyl(1-4C)alkyl, and each of R^(7b),R^(7c), R^(7d), R^(7e), R^(7f) and R^(7g) is independently hydrogen,(1-4C)alkyl, (3-6C)cycloalkyl, phenyl or phenyl(1-4C)alkyl, wherein eachphenyl group is unsubstituted or substituted by 1 or 2 substituentsindependently selected from halo, (1-4C)alkyl and (1-4C)alkoxy; and

R⁸ is selected from (1-4C)alkyl, (1-4C)alkyleneNR^(8a)R^(8b), andphenyl, each of R^(8a) and R^(8b) is independently hydrogen or(1-4C)alkyl; or R⁸ is taken together with R⁷ to form a ring having 1 to2 oxygen atoms, where said ring is unsubstituted or substituted by 1 or2 (1-4C)alkyl substituents;

wherein each alkyl and alkoxy group in R¹, R^(1a-1k), R², R^(2a-2k), R³,R⁵, R⁷, R^(7a-7g), R⁸, and R^(8a-8b) is optionally substituted with 1 to5 fluoro substituents;

or a pharmaceutically acceptable salt or solvate or stereoisomerthereof.

Another aspect of the invention pertains to a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a therapeuticallyeffective amount of a compound of formula I or a pharmaceuticallyacceptable salt or solvate or stereoisomer thereof. Yet another aspectof the invention pertains to compositions comprising a compound offormula I in combination with one or more other therapeutic agents.Accordingly, in one embodiment, the invention is directed to acomposition comprising (a) a pharmaceutically acceptable carrier and atherapeutically effective amount of a compound of formula I or apharmaceutically acceptable salt or solvate or stereoisomer thereof; and(b) a therapeutically effective amount of an agent selected from asteroidal anti-inflammatory agent such as a corticosteroid; a β₂adrenergic receptor agonist; a phosphodiesterase-4 inhibitor; or acombination thereof; wherein the compound of formula I and the agent areformulated together or separately. When the agent is formulatedseparately, a pharmaceutically acceptable carrier may be included.

Compounds of the invention possess muscarinic receptor antagonistactivity. Accordingly, compounds of formula I are expected to be usefulfor treating pulmonary disorders such as chronic obstructive pulmonarydisease and asthma.

Yet another aspect of the invention relates to a method for treating apulmonary disorder, comprising administering to a patient atherapeutically effective amount of a compound of formula I or apharmaceutically acceptable salt or solvate or stereoisomer thereof.

Still another aspect of the invention pertains to a method of producingbronchodilation in a patient, comprising administering to the patient abronchodilation-producing amount of a compound of formula I or apharmaceutically acceptable salt or solvate or stereoisomer thereof. Inone embodiment, the compound is administered by inhalation.

The invention is also directed to a method of treating chronicobstructive pulmonary disease or asthma, comprising administering to apatient a therapeutically effective amount of a compound of formula I ora pharmaceutically acceptable salt or solvate or stereoisomer thereof.

Another aspect of the invention relates to a method for antagonizing amuscarinic receptor in a mammal, comprising administering to the mammal,a therapeutically effective amount of the compound of formula I.

Since compounds of the invention possess muscarinic receptor antagonistactivity, such compounds are also useful as research tools. Accordingly,another aspect of the invention is directed to a method for using acompound of formula I or a pharmaceutically acceptable salt or solvateor stereoisomer thereof as a research tool for studying a biologicalsystem or sample, or for discovering new chemical compounds havingmuscarinic receptor antagonist activity.

The invention is also directed to processes and novel intermediatesuseful for preparing compounds of formula I, and pharmaceuticallyacceptable salts, solvates, and stereoisomers thereof. Accordingly,another aspect of the invention relates to a process of preparing acompound of formula I, comprising:

-   -   (a) reacting a compound of formula II with a compound of formula        III; or    -   (b) coupling a compound of formula IVa with a compound of        formula Va′ or Va″, or coupling a compound of formula IVb with a        compound of formula Vb′ or Vb″; or    -   (c) reacting a compound of formula VI with a compound of formula        VII; or    -   (d) reacting a compound of formula II with a compound of formula        VIII in the presence of a reducing agent; or    -   (e) reacting a compound of formula IX with a compound of formula        VII in the presence of a reducing agent;        and then removing any protecting groups, if necessary, to        provide a compound of formula I; wherein compounds of formula        I-IX are as defined herein.

In one embodiment, the above process further comprises the step offorming a pharmaceutically acceptable salt of a compound of formula I.In other embodiments, the invention is directed to the other processesdescribed herein; and to the product prepared by any of the processesdescribed herein.

The invention is also directed to a compound of formula I or apharmaceutically acceptable salt or solvate or stereoisomer thereof, foruse in therapy or as a medicament.

Additionally, the invention is directed to the use of a compound offormula I or a pharmaceutically acceptable salt or solvate orstereoisomer thereof, for the manufacture of a medicament; especiallyfor the manufacture of a medicament for the treatment of a pulmonarydisorder or for antagonizing a muscarinic receptor in a mammal.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to novel biphenyl compounds of formula I, andpharmaceutically acceptable salts, solvates or stereoisomers thereof.These compounds may contain one or more chiral centers and therefore,the invention is directed to racemic mixtures; pure stereoisomers (i.e.,enantiomers or diastereomers); stereoisomer-enriched mixtures and thelike unless otherwise indicated. When a particular stereoisomer is shownor named herein, it will be understood by those skilled in the art thatminor amounts of other stereoisomers may be present in the compositionsof the invention unless otherwise indicated, provided that the desiredutility of the composition as a whole is not eliminated by the presenceof such other isomers.

The compounds of formula I also contain several basic groups (e.g.,amino groups) and therefore, compounds of formula I can exist as thefree base or in various salt forms. All such salt forms are includedwithin the scope of the invention. Furthermore, solvates of compounds offormula I or salts thereof are included within the scope of theinvention.

Additionally, where applicable, all cis-trans or E/Z isomers (geometricisomers), tautomeric forms and topoisomeric forms of the compounds offormula I are included within the scope of the invention unlessotherwise specified.

The compounds of formula I, as well as those compounds used in theirsynthesis, may also include isotopically-labeled compounds, i.e., whereone or more atoms have been enriched with atoms having an atomic massdifferent from the atomic mass predominately found in nature. Examplesof isotopes that may be incorporated into the compounds of formula Iinclude, but are not limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O and ¹⁷O.

The nomenclature used herein to name the compounds of the invention isillustrated in the Examples herein. This nomenclature has been derivedusing the commercially-available AutoNom software (MDL, San Leandro,Calif.). For example, compounds of formula I wherein W is O havetypically been named as ester derivatives of biphenyl-2-ylcarbamic acid.

Representative Embodiments

The following substituents and values are intended to providerepresentative examples of various aspects and embodiments of theinvention. These representative values are intended to further defineand illustrate such aspects and embodiments and are not intended toexclude other embodiments or to limit the scope of the invention. Inthis regard, the representation that a particular value or substituentis preferred is not intended in any way to exclude other values orsubstituents from the invention unless specifically indicated.

The value for a is 0, 1, 2, 3, 4 or 5; particularly 0, 1 or 2, and evenmore particularly 0 or 1. The value for b is 0, 1, 2, 3 or 4;particularly 0, 1 or 2, and even more particularly 0 or 1. In oneembodiment, a is 0. In another embodiment, b is zero. In yet anotherembodiment, both a and b are 0.

When present, each R¹ may be at the 2, 3, 4, 5 or 6-position of thephenyl ring to which it is attached. Each R¹ is independently selectedfrom (1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl, (3-6C)cycloalkyl, cyano,halo, —OR^(1a), —C(O)OR^(1b), —SR^(1c), —S(O)R^(1d), —S(O)₂R^(1e),−NR^(1f)R^(1g), —NR^(1h)S(O)₂R^(1i), and —NR^(1j)C(O)R^(1k), examples ofwhich include methyl, fluoro, chloro, bromo, hydroxy, methoxy, amino,methylamino, dimethylamino and the like. In a particular embodiment,each R¹ is independently selected from (1-4C)alkyl, halo, —OR^(1a) and—NR^(1f)R^(1g). Particular values for R¹ are fluoro or chloro.

When present, each R² may be at the 3, 4, 5 or 6-position on thephenylene ring to which it is attached (where the carbon atom on thephenylene ring attached to the nitrogen atom is position 1). Each R² isindependently selected from (1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl,(3-6C)cycloalkyl, cyano, halo, —OR^(2a), —C(O)OR^(2b), —SR^(2c),—S(O)R^(2d), —S(O)₂R^(2e), —NR^(2f)R^(2g), —NR^(2h)S(O)₂R^(2i), and—NR^(2j)C(O)R^(2k), examples of which include methyl, fluoro, chloro,bromo, hydroxy, methoxy, amino, methylamino, dimethylamino and the like.In one embodiment, each R² is independently selected from (1-4C)alkyl,halo, —OR^(2a) and —NR^(2f)R^(2g). Particular values for R² are fluoroor chloro.

Each R^(1a-1k) and R^(2a-2k) group as used in R¹ and R², respectively,is independently hydrogen, (1-4C)alkyl or phenyl(1-4C)alkyl, examples ofwhich include hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl or benzyl. In one embodiment, thesegroups are independently hydrogen or (1-4C)alkyl. In another embodiment,these groups are independently hydrogen, methyl or ethyl. In addition,each alkyl and alkoxy group in R¹, R^(1a-1k), R², and R^(2a-2k) isoptionally substituted with 1 to 5 fluoro substituents.

W can be O or NW^(a). Generally, it has been found that compounds inwhich W represents O exhibit particularly high affinity for muscarinicreceptors. Accordingly, in a particular embodiment of the invention, Wrepresents O.

When W is NW^(a), W^(a) is hydrogen or (1-4C)alkyl, examples of whichinclude hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl and tert-butyl. In one embodiment, W^(a) is hydrogenor (1-3C)alkyl. In another embodiment, W^(a) is hydrogen, methyl orethyl, particularly hydrogen or methyl. In yet another embodiment, W^(a)is hydrogen.

The value for c is 0, 1, 2, 3, 4, or 5; particularly 0, 1, or 2; andmore particularly 0 or 1. In one particular embodiment, c is 0.

In one embodiment, each R³ is at the 3, 4 or 5-position on thepiperidine ring (where the nitrogen atom of the piperidine ring isposition 1). In a particular embodiment, R³ is at 4-position on thepiperidine ring. In another embodiment, R³ is at the 1-position of thepiperidine ring, i.e., on the nitrogen atom of the piperidine ring thusforming a quaternary amine salt. Each R³ is independently (1-4C)alkyl,or two R³ groups are joined to form (1-3C)alkylene, (2-3C)alkenylene oroxiran-2,3-diyl. In one embodiment, each R³ is independently (1-4C)alkylsuch as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyland tert-butyl. In addition, each alkyl group in R³ is optionallysubstituted with 1 to 5 fluoro substituents. In one embodiment, each R³is independently (1-4C) alkyl, and in another embodiment, each R³ isindependently methyl or ethyl.

In yet another embodiment, two R³ groups are joined to form a(1-3C)alkylene or (2-3C)alkenylene group. For example, two R³ groups atthe 2 and 6-positions on the piperidine ring can be joined to form anethylene bridge (i.e., the piperidine ring and the R³ groups form an8-azabicyclo[3.2.1]octane ring); or two R³ groups at the 1 and4-positions on the piperidine ring can be joined to form an ethylenebridge (i.e., the piperidine ring and the R³ groups form an1-azabicyclo[2.2.2]octane ring). In this embodiment, other R³ groups asdefined herein may also be present.

In still another embodiment, two R³ groups are joined to form aoxiran-2,3-diyl group. For example, two R³ groups at the 2 and6-positions on the piperidine ring can be joined to form a3-oxatricyclo[3.3.1.0^(2,4)]nonane ring). In this embodiment, other R³groups as defined herein may also be present.

A is selected from:

In one particular embodiment, A is

In another particular embodiment, A is

In yet another particular embodiment, A is

The value for m is 0 or 1. In one embodiment, m is 0.

The value for r is 2, 3, or 4. In one embodiment, r is 3.

The value for s is 0, 1 or 2. A particular value for s is 0.

The value for t is 0, 1 or 2. A particular value for t is 1.

R⁴ represents hydrogen, (1-4C)alkyl, or (3-4C)cycloalkyl. Examples of(1-4C)alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl and tert-butyl. Examples of (3-4C)cycloalkyl groupsinclude cyclopropyl and cyclobutyl. In one embodiment, R⁴ representshydrogen or (1-4C)alkyl. In a particular embodiment, R⁴ is hydrogen ormethyl. In another embodiment, R⁴ is hydrogen.

Ar¹ is a phenylene group or a (3-5C)heteroarylene group containing 1 or2 heteroatoms independently selected from oxygen, nitrogen or sulfur.The value for q is 0, 1, 2, 3, or 4, particularly 0, 1, 2 or 3. In oneembodiment, q is 0, 1 or 2. Thus, the phenylene or heteroarylene groupmay be unsubstituted (q is 0) or substituted with 1 to 4 R⁵substituents, which are independently selected from halo, hydroxy,(1-4C)alkyl and (1-4C)alkoxy. In addition, each alkyl and alkoxy groupin R⁵ is optionally substituted with 1 to 5 fluoro substituents. Thepoint of attachment for Ar¹ is at any available carbon or heteroatomring atom. In certain embodiments, Ar¹ is a phenylene group attached atthe meta or para position.

In one embodiment Ar¹ is phen-1,3-ylene or phen-1,4-ylene wherein thephenylene group is unsubstituted or substituted with 1, 2 or 3 R⁵substituents. Representative R⁵ substituents include fluoro, chloro,bromo, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, methoxy, ethoxy, isopropoxy, difluoromethyl,trifluoromethyl, 2,2,2-trifluoroethyl and trifluoromethoxy. Particularexamples of Ar¹ groups in this embodiment include2-fluorophen-1,4-ylene, 3-fluorophen-1,4-ylene, 2-chlorophen-1,4-ylene,3-chlorophen-1,4-ylene, 2-methylphen-1,4-ylene, 3-methylphen-1,4-ylene,2-methoxyphen-1,4-ylene, 3-methoxyphen-1,4-ylene,2-trifluoromethoxyphen-1,4-ylene, 3-trifluoromethoxyphen-1,4-ylene,2,3-difluorophen-1,4-ylene, 2,5-difluorophen-1,4-ylene,2,6-difluorophen-1,4-ylene, 2,3-dichlorophen-1,4-ylene,2,5-dichlorophen-1,4-ylene, 2,6-dichlorophen-1,4-ylene,2-chloro-5-methoxyphen-1,4-ylene, 2-chloro-6-methoxyphen-1,4-ylene,2-chloro-5-trifluoromethoxyphen-1,4-ylene,2-chloro-6-trifluoromethoxyphen-1,4-ylene, and2,5-dibromophen-1,4-ylene.

In another embodiment, Ar¹ is a (3-5C)heteroarylene group containing 1or 2 heteroatoms independently selected from oxygen, nitrogen andsulfur; wherein the heteroarylene group is unsubstituted or substitutedwith 1 or 2 R⁵ substituents. Representative heteroarylene groups includedivalent species of pyrrole, imidazole, thiazole, oxazole, furan,thiophene, pyrazole, isoxazole, isothiazole, pyridine, pyrazine,pyridazine and pyrimidine, where the point of attachment is at anyavailable carbon or nitrogen ring atom. More specific examples of suchAr¹ groups include 2,5-furylene, 2,4-thienylene, 2,5-thienylene,2,5-pyridylene, 2,6-pyridylene, and 2,5-pyrrolylene. Representative R⁵substituents include fluoro, chloro, methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, methoxy, ethoxy, isopropoxy,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl andtrifluoromethoxy. Particular examples of substituted Ar¹ groups include3-fluoro-2,5-thienylene, 3-chloro-2,5-thienylene,3-methyl-2,5-thienylene, 3-methoxy-2,5-thienylene, and3-methoxy-6-chloro-2,5-pyridylene.

In one particular embodiment, Ar¹ represents phen-1,3-ylene,phen-1,4-ylene, 2,4-thienylene or 2,5-thienylene; wherein the phenyleneor thienylene group is optionally substituted with 1 or 2 R⁵substituents. In another particular embodiment, Ar¹ representsphen-1,4-ylene or 2,4-thienylene optionally substituted with 1 or 2 R⁵substituents.

R⁶ represents hydrogen, (1-4C)alkyl, (3-4C)cycloalkyl, —C(O)(1-4C)alkyl,(0-3C)alkyleneC(O)OR^(6a), —C(O)heterocyclyl,—C(O)CH(NH₂)(1-4C)alkyleneQ, -(1-4C)alkyleneC(O)Z, —C(O)(1-4C)alkyleneZ,or —S(O)₂(1-4C)alkyleneZ. Q is a nitrogen-containing substituentselected from —NR^(6b)R^(6c) and heteroaryl. Z is a nitrogen-containingsubstituent selected from —NR^(6d)R^(6e) and heterocyclyl. R^(6a) ishydrogen or (1-4C)alkyl. Each of R^(6b), R^(6c), R^(6d) and R^(6e)independently represents hydrogen, (1-4C)alkyl, (3-6C)cycloalkyl orhydroxyphenyl, and (1-4C)alkyl is unsubstituted or substituted by 1 or 2substituents independently selected from amido, cyano, furyl, hydroxyl,and methylimidazolyl. The heterocyclyl contains 1 or 2 nitrogen atoms,and is unsubstituted or substituted by 1 or 2 substituents independentlyselected from hydroxyl, amido, (1-4C)alkoxy, oxo, —S(O)₂(1-4C)alkyl,—(CH₂)O(1-4C)alkyl, -(1-4C)alkyleneOH, —NR^(6f)R^(6g) and—C(O)NR^(6h)R^(6i), where each of R^(6f), R^(6g) R^(6h) and R^(6i)independently represents hydrogen or (1-4C)alkyl. The heteroarylcontains 1 or 2 nitrogen atoms. The heterocyclyl and heteroaryl groupsmay contain other heteroatoms, in addition to the 1 or 2 nitrogen atoms.For example the heterocyclyl can be a morpholinyl group.

In one embodiment, R⁶ represents hydrogen, (1-4C)alkyl, or(3-4C)cycloalkyl, examples of which include methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclopropyl andcyclobutyl. In another embodiment, R⁶ represents hydrogen or(1-4C)alkyl, particularly methyl. In another particular embodiment, R⁶is methyl. In yet another embodiment, R⁶ is hydrogen.

X¹ is selected from (1-3C)alkylene, —C(O)(1-3C)alkylene,(1-3C)alkyleneC(O)—, —SO₂—, —SO₂(1-3C)alkylene and (1-3C)alkyleneSO₂—.The alkylene group in any X¹ is optionally substituted with 1 or 2substituents independently selected from (1-4C)alkyl and —NR^(Xa)R^(Xb),where R^(Xa) and R^(Xb) are independently selected from hydrogen and(1-4alkyl). In one embodiment, X¹ is selected from (1-3C)alkylene,—C(O)(1-3C)alkylene, (1-3C)alkyleneC(O)— or —SO₂—. In anotherembodiment, X¹ is (1-3C)alkylene such as —CH₂— and —(CH₂)₂—. In yetanother embodiment, X¹ is —SO₂—.

The value for p is 0, 1, or 2. Particular values for p are 0 or 1. Inone embodiment, p is 0. In another embodiment, p is 1.

Each R⁷ is independently (1-4C)alkyl, (2-4C)alkenyl, (2-4C)alkynyl,(3-6C)cycloalkyl, cyano, nitro, halo,N,N-di(1-4C)alkylamino(2-4C)alkoxy, —OR^(7a), —C(O)OR^(7b), —SR^(7c),—S(O)R^(7d), —S(O)₂R^(7e) or —NR^(7f)R^(7g). R^(7a) is (1-4C)alkyl,(3-6C)cycloalkyl, phenyl or phenyl(1-4C)alkyl. Each R^(7b), R^(7c),R^(7d), R^(7e), R^(7f) and R^(7g) as used in R⁷ is independentlyhydrogen, (1-4C)alkyl, (3-6C)cycloalkyl, phenyl or phenyl(1-4C)alkyl.Each phenyl group in R^(7a-7g) is unsubstituted or substituted by 1 or 2substituents independently selected from halo, (1-4C)alkyl and(1-4C)alkoxy. In addition, each alkyl and alkoxy group in R⁷ andR^(7a-7g) is optionally substituted with 1 to 5 fluoro substituents. Inone embodiment, each R⁷ independently represents halo, (1-4C)alkyl, or(1-4C)alkoxy, where the alkyl and alkoxy groups are optionallysubstituted with 1 to 3 fluoro substituents. In another embodiment, eachR⁷ is independently selected from fluoro, chloro, bromo, methyl,methoxy, trifluoromethyl or trifluoromethoxy. In another embodiment,each R⁷ is —OR^(7a), where R^(7a) is (1-4C)alkyl such as methyl or(3-6C)cycloalkyl such as cyclopentyl.

R⁸ is selected from (1-4C)alkyl, (1-4C)alkyleneNR^(8a)R^(8b), andphenyl; or R⁸ is taken together with R⁷ to form a ring having 1 to 2oxygen atoms, where the ring is unsubstituted or substituted by 1 or 2(1-4C)alkyl substituents. Each of R^(8a) and R^(8b) is independentlyhydrogen or (1-4C)alkyl. In addition, each alkyl group in R⁸ andR^(8a-8b) is optionally substituted with 1 to 5 fluoro substituents. Inone embodiment, R⁸ is (1-4C)alkyl such as methyl or methyl substitutedwith 2 or 3 fluoro substituents. In another embodiment, R⁸ is(1-4C)alkyleneNR^(8a)R^(8b), where each of R^(8a) and R^(8b) isindependently (1-4C)alkyl such as methyl or ethyl, an example of whichis —(CH₂)₃N(CH₃)₂. In a particular embodiment, R⁸ is phenyl. In oneembodiment, R⁸ is taken together with R⁷ to form a ring having 1 to 2oxygen atoms, and the ring is unsubstituted or substituted by 1 or 2(1-4C)alkyl substituents. Exemplary R⁸/R⁷ chains include, —O(CH₂)—,—(CH₂)₂—C(CH₃)₂—, —(CH₂)₂—, and —O(CH₂)₂—.

As noted in formula I, the —OR⁸ group can be located at the ortho, metaor para position. In one embodiment, the —OR⁸ group is located at themeta or para position; and in a particular embodiment, the —OR⁸ group islocated at the para position.

A particular group of compounds of interest are compounds of formula Iwherein a, b and c are 0. Another particular group of compounds ofinterest are compounds of formula I wherein W represents O. Anotherparticular group of compounds of interest are compounds of formula Iwherein m is 0. Other compounds of interest are those where p is 1 andR⁷ is —OR^(7a), where R^(7a) is (1-4C)alkyl or (3-6C)cycloalkyl. Othercompounds of interest are those where R⁶ is hydrogen or methyl.Particular group of compounds of interest are compounds of formula Iwherein X¹ is (1-3C)alkylene or —SO₂—.

Combinations of the foregoing are also of interest. For example, onegroup of compounds of interest are compounds of formula I having any ofthe aforementioned substituents, and where A is

m is 0, and r is 3. Another group of compounds of interest are thosehaving any of the aforementioned substituents, and where A is

m is 0, s is 0, t is 1, and Ar¹ is phenylene. Another group of compoundsof interest are those having any of the aforementioned substituents, andwhere A is

m is 0, s is 0, t is 1, and Ar¹ is phenylene.

Another group of compounds of interest are those of formula Ia:

wherein p, A, X¹, R⁶, R⁷, and R⁸ are as defined above.

In addition, particular compounds of formula I that are of interestinclude:

biphenyl-2-ylcarbamic acid 1-(2-{5-[4-(3-dimethylaminopropoxy)benzylamino]pentylcarbanoyl}ethyl)piperidin-4-yl ester;

biphenyl-2-ylcarbamic acid1-{2-[5-(4-methoxybenzylamino)pentylcarbamoyl]ethyl}piperidin-4-ylester;

biphenyl-2-ylcarbamic acid1-{2-[5-(3-cyclopentyloxy-4-methoxybenzylamino)pentylcarbamoyl]ethyl}piperidin-4-ylester;

biphenyl-2-ylcarbamic acid1-{2-[5-(3,4-dimethoxybenzylamino)pentylcarbamoyl]ethyl}piperidin-4-ylester;

biphenyl-2-ylcarbamic acid1-{2-[5-(4-trifluoromethoxybenzylamino)pentylcarbamoyl]ethyl}piperidin-4-ylester;

biphenyl-2-ylcarbamic acid1-{2-[5-(4-difluoromethoxybenzylamino)pentylcarbamoyl]ethyl}piperidin-4-ylester;

biphenyl-2-ylcarbamic acid1-{2-[5-(4-phenoxybenzylamino)pentylcarbamoyl]-ethyl}piperidin-4-ylester;

biphenyl-2-ylcarbamic acid1-(2-{5-[(benzo[1,3]dioxol-5-ylmethyl)amino]pentylcarbamoyl}ethyl)piperidin-4-ylester;

biphenyl-2-ylcarbamic acid1-(2-{5-[(2,2-dimethylchroman-6-ylmethyl)amino]pentylcarbamoyl}ethyl)piperidin-4-ylester;

biphenyl-2-ylcarbamic acid1-(2-{5-[(2,3-dihydrobenzofuran-5-ylmethyl)amino]pentylcarbamoyl}ethyl)piperidin-4-ylester;

biphenyl-2-ylcarbamic acid1-(2-{5-[(2,3-dihydro-benzo[1,4]dioxin-6-ylmethyl)amino]pentylcarbamoyl}ethyl)piperidin-4-ylester;

biphenyl-2-ylcarbamic acid1-{2-[5-(4-methoxybenzenesulfonylamino)pentylcarbamoyl]ethyl}piperidin-4-ylester;

biphenyl-2-ylcarbamic acid1-(2-{5-[2-(4-methoxyphenyl)ethylamino]pentylcarbamoyl}ethyl)piperidin-4-ylester;

biphenyl-2-ylcarbamic acid1-(2-{5-[2-(3,4-dimethoxyphenyl)ethylamino]pentylcarbamoyl}ethyl)piperidin-4-ylester;

biphenyl-2-ylcarbamic acid1-[2-(4-{[4-(3-dimethylaminopropoxy)benzylamino]methyl}phenylcarbamoyl)ethyl]piperidin-4-ylester;

biphenyl-2-ylcarbamic acid1-(2-{4-[(4-trifluoromethoxybenzylamino)methyl]phenylcarbamoyl}ethyl)piperidin-4-ylester;

biphenyl-2-ylcarbamic acid1-(2-{4-[(4-difluoromethoxybenzylamino)methyl]phenylcarbamoyl}ethyl)piperidin-4-ylester;

biphenyl-2-ylcarbamic acid1-(2-{4-[(3-cyclopentyloxy-4-methoxybenzylamino)methyl]phenylcarbamoyl}ethyl)piperidin-4-ylester;

biphenyl-2-ylcarbamic acid1-(2-{4-[(4-methoxybenzylamino)methyl]phenylcarbamoyl}ethyl)piperidin-4-ylester;

biphenyl-2-ylcarbamic acid1-[2-(4-{[(benzo[1,3]dioxol-5-ylmethyl)amino]methyl}phenylcarbamoyl)ethyl]piperidin-4-ylester;

biphenyl-2-ylcarbamic acid1-[2-({4-[(4-methoxybenzylamino)methyl]benzoyl}methylamino)ethyl]piperidin-4-ylester; and

biphenyl-2-ylcarbamic acid1-[2-({4-[(2-methoxybenzylamino)methyl]benzoyl}methylamino)ethyl]piperidin-4-ylester;

or a pharmaceutically acceptable salt or solvate thereof.

DEFINITIONS

When describing the compounds, compositions, methods and processes ofthe invention, the following terms have the following meanings unlessotherwise indicated.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to 10 carbon atoms. Representative alkyl groupsinclude, by way of example, methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl,n-nonyl, n-decyl and the like.

The term “alkylene” means a divalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from 1 to 10 carbon atoms. Representativealkylene groups include, by way of example, methylene, ethane-1,2-diyl(“ethylene”), propane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl,pentane-1,5-diyl and the like.

The term “alkoxy” means a monovalent group of the formula (alkyl)-O—,where alkyl is as defined herein. Representative alkoxy groups include,by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,sec-butoxy, isobutoxy, tert-butoxy and the like.

The term “alkenyl” means a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon double bonds. Unless otherwisedefined, such alkenyl groups typically contain from 2 to 10 carbonatoms. Representative alkenyl groups include, by way of example,ethenyl, n-propenyl, isopropenyl, n-but-2-enyl, n-hex-3-enyl and thelike. The term “alkenylene” means a divalent alkenyl group.

The term “alkynyl” means a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon triple bonds. Unless otherwisedefined, such alkynyl groups typically contain from 2 to 10 carbonatoms. Representative alkynyl groups include, by way of example,ethynyl, n-propynyl, n-but-2-ynyl, n-hex-3-ynyl and the like. The term“alkynylene” means a divalent alkynyl group.

The term “aryl” means a monovalent aromatic hydrocarbon having a singlering (i.e., phenyl) or fused rings (i.e., naphthalene). Unless otherwisedefined, such aryl groups typically contain from 6 to 10 carbon ringatoms. Representative aryl groups include, by way of example, phenyl andnaphthalene-1-yl, naphthalene-2-yl, and the like. The term “arylene”means a divalent aryl group.

The term “azacycloalkyl” means a monovalent heterocyclic ring containingone nitrogen atom, i.e., a cycloalkyl group in which one carbon atom hasbeen replaced with a nitrogen atom. Unless otherwise defined, suchazacycloalkyl groups typically contain from 2 to 9 carbon atoms.Representative examples of an azacycloalkyl group are pyrrolidinyl andpiperidinyl groups. The term “azacycloalkylene” means a divalentazacycloakyl group. Representative examples of an azacycloalkylene groupare pyrrolidinylene and piperidinylene groups.

The term “cycloalkyl” means a monovalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkyl groupstypically contain from 3 to 10 carbon ring atoms. Representativecycloalkyl groups include, by way of example, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl and the like. The term “cycloalkylene” means adivalent cycloalkyl group.

The term “halo” means fluoro, chloro, bromo and iodo.

The term “heteroaryl” means a monovalent aromatic group having a singlering or two fused rings and containing in the ring at least oneheteroatom (typically 1 to 3 heteroatoms) selected from nitrogen, oxygenand sulfur. Unless otherwise defined, such heteroaryl groups typicallycontain from 5 to 10 total ring atoms. Representative heteroaryl groupsinclude, by way of example, monovalent species of pyrrole, imidazole,thiazole, oxazole, furan, thiophene, triazole, pyrazole, isoxazole,isothiazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine,indole, benzofuran, benzothiophene, benzimidazole, benzthiazole,quinoline, isoquinoline, quinazoline, quinoxaline and the like, wherethe point of attachment is at any available carbon or nitrogen ringatom. The term “heteroarylene” means a divalent heteroaryl group.

The term “heterocyclyl” or “heterocyclic” means a monovalent saturatedor unsaturated (non-aromatic) group having a single ring or multiplecondensed rings and containing in the ring at least one heteroatom(typically 1 to 3 heteroatoms) selected from nitrogen, oxygen andsulfur. Unless otherwise defined, such heterocyclic groups typicallycontain from 2 to 9 total ring carbon atoms. Representative heterocyclicgroups include, by way of example, monovalent species of pyrrolidine,imidazolidine, pyrazolidine, piperidine, 1,4-dioxane, morpholine,thiomorpholine, piperazine, 3-pyrroline and the like, where the point ofattachment is at any available carbon or nitrogen ring atom. The term“heterocyclene” means a divalent heterocyclyl or heterocyclic group.

When a specific number of carbon atoms is intended for a particular termused herein, the number of carbon atoms is shown in parenthesespreceding the term. For example, the term “(1-4C)alkyl” means an alkylgroup having from 1 to 4 carbon atoms.

The term “pharmaceutically acceptable salt” means a salt which isacceptable for administration to a patient such as a mammal (e.g., saltshaving acceptable mammalian safety for a given dosage regime). Suchsalts can be derived from pharmaceutically acceptable inorganic ororganic bases and from pharmaceutically acceptable inorganic or organicacids. Salts derived from pharmaceutically acceptable inorganic basesinclude ammonium, calcium, copper, ferric, ferrous, lithium, magnesium,manganic, manganous, potassium, sodium, zinc and the like. Particularlypreferred are ammonium, calcium, magnesium, potassium and sodium salts.Salts derived from pharmaceutically acceptable organic bases includesalts of primary, secondary and tertiary amines, including substitutedamines, cyclic amines, naturally-occurring amines and the like such asarginine, betaine, caffeine, choline, N,N′-dibenzylethylenediamine,diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol,ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine,glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. Salts derived frompharmaceutically acceptable acids include acetic, ascorbic,benzenesulfonic, benzoic, camphosulfonic, citric, ethanesulfonic,edisylic, fumaric, gentisic, gluconic, glucoronic, glutamic, hippuric,hydrobromic, hydrochloric, isethionic, lactic, lactobionic, maleic,malic, mandelic, methanesulfonic, mucic, naphthalenesulfonic,naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic, nicotinic,nitric, orotic, pamoic, pantothenic, phosphoric, succinic, sulfuric,tartaric, p-toluenesulfonic, xinafoic and the like. Particularlypreferred are citric, hydrobromic, hydrochloric, isethionic, maleic,naphthalene-1,5-disulfonic, phosphoric, sulfuric and tartaric acids.

The term “salt thereof” means a compound formed when the hydrogen of anacid is replaced by a cation such as a metal cation or an organic cationand the like. Preferably, the salt is a pharmaceutically acceptablesalt. This is not required however, since some salts (e.g., salts ofintermediate compounds) are not intended to be administered to patients.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, i.e. a compound of formula I or apharmaceutically acceptable salt thereof, and one or more molecules of asolvent. Such solvates are typically crystalline solids having asubstantially fixed molar ratio of solute and solvent. Representativesolvents include, by way of example, water, methanol, ethanol,isopropanol, acetic acid and the like. When the solvent is water, thesolvate formed is a hydrate.

It will be appreciated that the term “or a pharmaceutically acceptablesalt or solvate or stereoisomer thereof” is intended to include allpermutations of salts, solvates and stereoisomers such as a solvate of apharmaceutically acceptable salt of a stereoisomer of a compound offormula I.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.For example, a therapeutically effective amount for antagonizing amuscarinic receptor is that amount which will achieve the desiredantagonizing effect. Similarly, a therapeutically effective amount fortreating a pulmonary disorder is that amount that will achieve thedesired therapeutic result, which may be disease prevention,amelioration, suppression or alleviation, as described below.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as COPD) in a patientsuch as a mammal (particularly a human) that includes:

-   -   (a) preventing the disease or medical condition from occurring,        i.e., prophylactic treatment of a patient believed to be at risk        of contracting or being pre-disposed to such disease or medical        condition;    -   (b) ameliorating the disease or medical condition, i.e.,        eliminating or causing regression of the disease or medical        condition in a patient having such disease or medical condition;    -   (c) suppressing the disease or medical condition, i.e., slowing        or arresting the development of the disease or medical condition        in a patient having such disease or medical condition; or    -   (d) alleviating the symptoms of the disease or medical condition        in a patient having such disease or medical condition.

The term “unit dosage form” refers to a physically discrete unitsuitable for dosing a patient, i.e., each unit containing apredetermined quantity of a compound of the invention calculated toproduce the desired therapeutic effect either alone or in combinationwith one or more additional units. For example, such unit dosage formsmay be capsules, tablets, pills, and the like.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise undesirable. For example, the term“pharmaceutically acceptable carrier” refers to a material that can beincorporated into a composition and administered to a patient withoutcausing undesirable biological effects or interacting in a deleteriousmanner with other components of the composition. Such pharmaceuticallyacceptable materials typically have met the required standards oftoxicological and manufacturing testing, and include those materialsidentified as suitable inactive ingredients by the U.S. Food and Drugadministration.

The term “leaving group” means a functional group or atom which can bedisplaced by another functional group or atom in a substitution reactionsuch as a nucleophilic substitution reaction. By way of example,representative leaving groups include chloro, bromo and iodo groups;sulfonic ester groups such as mesylate, tosylate, brosylate, nosylateand the like; and acyloxy groups such as acetoxy, trifluoroacetoxy andthe like.

The term “protected derivatives thereof” means a derivative of thespecified compound in which one or more functional groups of thecompound are protected from undesired reactions with a protecting orblocking group. Functional groups which may be protected include, by wayof example, carboxylic acid groups, amino groups, hydroxyl groups, thiolgroups, carbonyl groups and the like. Representative protecting groupsfor carboxylic acids include esters (such as a p-methoxybenzyl ester),amides and hydrazides; for amino groups, carbamates (such astert-butoxycarbonyl) and amides; for hydroxyl groups, ethers and esters;for thiol groups, thioethers and thioesters; for carbonyl groups,acetals and ketals; and the like. Such protecting groups are well-knownto those skilled in the art and are described, for example, in T. W.Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, ThirdEdition, Wiley, N.Y., 1999, and references cited therein.

The term “amino-protecting group” means a protecting group suitable forpreventing undesired reactions at an amino group. Representativeamino-protecting groups include, but are not limited to,tert-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl (Cbz),9-fluorenylmethoxycarbonyl (Fmoc), formyl, trimethylsilyl (TMS),tert-butyldimethylsilyl (TBS), and the like.

The term “carboxy-protecting group” means a protecting group suitablefor preventing undesired reactions at a carboxy group. Representativecarboxy-protecting groups include, but are not limited to, esters suchas methyl, ethyl, tert-butyl, benzyl (Bn), p-methoxybenzyl (PMB),9-fluroenylmethyl (Fm), trimethylsilyl (TMS), tert-butyldimethylsilyl(TBS), diphenylmethyl (benzhydryl, DPM) and the like.

The term “hydroxyl-protecting group” means a protecting group suitablefor preventing undesirable reactions at a hydroxyl group. Representativehydroxyl-protecting groups include, but are not limited to, silyl groupsincluding tri(1-6C)alkylsilyl groups such as trimethylsilyl (TMS),triethylsilyl (TES), tert-butyldimethylsilyl (TBS) and the like; esters(acyl groups) including (1-6C)alkanoyl groups such as formyl, acetyl andthe like; arylmethyl groups such as benzyl (Bn), p-methoxybenzyl (PMB),9-fluorenylmethyl (Fm), diphenylmethyl (benzhydryl, DPM) and the like.Additionally, two hydroxyl groups can also be protected as an alkylidenegroup such as prop-2-ylidine, formed, for example, by reaction with aketone such as acetone.

General Synthetic Procedures

The biphenyl compounds of the invention can be prepared from readilyavailable starting materials using the following general methods, theprocedures set forth in the Examples, or by using other methods,reagents, and starting materials that are readily available to those ofordinary skill in the art. Although a particular embodiment of thepresent invention may be shown or described herein, those skilled in theart will recognize that all embodiments or aspects of the presentinvention can be readily prepared. It will also be appreciated thatwhere typical or preferred process conditions (i.e., reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. While the optimum reaction conditions may varydepending on the particular reactants or solvent used, such conditionscan be readily determined by one skilled in the art by routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Thechoice of a suitable protecting group for a particular functional groupas well as suitable conditions for protection and deprotection of suchfunctional groups are well-known in the art. Protecting groups otherthan those illustrated in the procedures described herein may be used,if desired. For example, numerous protecting groups, and theirintroduction and removal, are described in T. W. Greene and G. M. Wuts,Protecting Groups in Organic Synthesis, Third Edition, Wiley, N.Y.,1999, and references cited therein.

By way of illustration, the compounds of formula I can be prepared by aprocess comprising:

(a) reacting a compound of formula II:

or a salt thereof, with a compound of formula III:

wherein L¹ represents a leaving group; or

(b) coupling a compound of formula IVa:

or a reactive derivative thereof; with a compound of formula Va′ or Va″:

or coupling a compound of formula IVb:

with a compound of formula Vb′ or Vb″:

or a reactive derivative thereof; or

(c) reacting a compound of formula VI:

wherein L⁵ represents a leaving group; with a compound of formula VII:

or

(d) reacting a compound of formula II with a compound of formula VIII:

(where “A” has one less carbon, i.e., m-1 instead of m) in the presenceof a reducing agent; or

(e) reacting a compound of formula IX:

(where “A” has one less carbon, i.e., r-1 instead of r, or t-1 insteadof t) with a compound of formula VII in the presence of a reducingagent; and

(f) removing any protecting groups that may be present to provide acompound of formula I; and optionally, forming a pharmaceuticallyacceptable salt thereof.

In one embodiment of the aforementioned process, a compound of formula Iis first synthesized with R⁶ being hydrogen, i.e., R⁶ is hydrogen in thecompound of formula III, Va′, Va″, Vb′, Vb″, VII, or VIII. Thisresulting compound can then be reacted further to replace this hydrogenwith an R⁶ substituent selected from (1-4C)alkyl, (3-4C)cycloalkyl,—C(O)(1-4C)alkyl, -(1-4C)alkyleneC(O)OR^(6a), —C(O)heterocyclyl,—C(O)CH(NH₂)(1-4C)alkyleneQ, -(1-4C)alkyleneC(O)Z, —C(O)(1-4C)alkyleneZ,and —S(O)₂(1-4C)alkyleneZ.

Generally, if a salt of one of the starting materials is used in theprocesses described above, such as an acid addition salt, the salt istypically neutralized before or during the reaction process. Thisneutralization reaction is typically accomplished by contacting the saltwith one molar equivalent of a base for each molar equivalent of acidaddition salt.

In process (a), the reaction between the compounds of formula II andIII, the leaving represented by L¹ can be, for example, a halo groupsuch as chloro, bromo or iodo, or a sulfonic ester group such asmesylate or tosylate. The reaction is conveniently performed in thepresence of a base, for example, a tertiary amine such asdiisopropylethylamine. Convenient solvents include nitriles such asacetonitrile. The reaction is conveniently conducted at a temperature inthe range of from 0 to 100° C.

Compounds of formula II are generally known in the art, or can beprepared by deprotecting a compound of formula X:

where P¹ represents an amino-protecting group such as a benzyl group.Benzyl groups are conveniently removed by reduction, using a hydrogen orammonium formate and a Group VIII metal catalyst such as palladium. WhenW represents NW^(a), the hydrogenation reaction is convenientlyperformed using Pearlman's catalyst (Pd(OH)₂).

Compounds of formula X can be prepared by reacting an isocyanatecompound of formula XI:

with a compound of formula XII:

Compounds of formula III can be prepared starting from a correspondingcompound in which L¹ represents a hydroxyl group, for example, byreaction of a halogenating agent such as thionyl chloride, to afford acompound of formula III in which L¹ represents halo such as chloro.Compounds of formula I, in which L¹ represents a hydroxyl group and Ahas the formula:

may be prepared, for example, by reacting a compound of formula Va′ orVa″ with an appropriate lactone such as γ-butyrolactone. Compounds offormula I, in which L¹ represents a hydroxyl group and A has theformula:

may be prepared, for example, by reacting a compound of formula Vb′ orVb″ with an appropriate amino-substituted alcohol such as 2-aminoethanolor 3-aminopropan-1-ol.

In process (b), a compound of formula IVa or reactive derivative thereofis reacted with a compound of formula Va′ or Va″, or a compound offormula IVb is reacted with a compound of formula Vb′ or Vb″ or reactivederivative thereof. By “reactive derivative” of compound IVa, Vb′, orVb″, it is meant that the carboxylic acid is activated, for example, byforming an anhydride or carboxylic acid halide such as a carboxylic acidchloride. Alternatively, the carboxylic acid can be activated usingconventional carboxylic acid/amine coupling reagents, suchcarbodiimides, O-(7-azabenzotriazol-1-yl-N,N,N′,N′ tetramethyluroniumhexafluorophosphate (HATU) and the like. This reaction is convenientlyperformed under conventional amide bond-forming conditions. The processis conveniently conducted at a temperature in the range of from −10 to100° C.

Compounds of formula IVa can be prepared by reacting a compound offormula II with a compound of formula XIIIa:L²-CH₂(CH₂)_(m)CH₂COOP²   XIIIawhere L² represents a leaving group including, for example, a halo groupsuch as chloro, bromo or iodo, or a sulfonic ester group such asmesylate or tosylate; and P² represents a hydrogen atom or acarboxyl-protecting group such as a (1-4C)alkyl group. If necessary, thecarboxyl-protecting group P², is then removed, for example, byhydrolysis under conventional conditions, such as by using lithiumhydroxide. Alternatively, when m is 0, compounds of formula IVa can beprepared by reacting II with CH₂═CHC(O)OP² and then removing thecarboxyl-protecting group P², if necessary.

Compounds of formula IVb can be prepared by reacting a compound offormula II with a compound of formula XIIIb:OHC(CH₂)_(m)CH₂NR⁴P³   XIIIbwhere P³ represents hydrogen or an amino-protecting group, such asbenzyl, in the presence of a reducing agent, such as sodiumtriacetoxyborohydride, followed if necessary by removing theamino-protecting group P³ by, for example, hydrogenation in the presenceof palladium.

Compounds of formula Va′ can be prepared by reacting a compound offormula VII with a compound of formula XIVa:

where P⁴ represents hydrogen or an amino-protecting group such astert-butoxycarbonyl, and L³ represents a leaving group including, forexample, a halo group such as chloro, bromo or iodo, or a sulfonic estergroup such as mesylate or tosylate; followed if necessary, by removingan amino-protecting group P⁴. Compound Va″ can be made in a similarmanner. Alternatively, such compounds can be prepared by reductiveamination of a compound of formula XVa:

using a compound of formula VII under conventional reaction conditionssuch as those described for processes (d) and (e).

Compounds of formula Vb′ can be prepared by reacting a compound offormula VII with a compound of formula XIVb:

where P⁵ represents hydrogen or a carboxyl-protecting group such asmethyl or ethyl, and L⁴ represents a leaving group, followed ifnecessary by removing the carboxyl protecting group P⁵. Compound Vb″ canbe made in a similar manner. Alternatively, such compounds can beprepared by reductive amination of a compound of formula XVb:

with a compound of formula VII under conventional reaction conditionssuch as those described for processes (d) and (e).

Referring to process (c), the leaving group represented by L⁵ can be,for example, a halo group such as chloro, bromo or iodo, or a sulfonicester group such as mesylate or tosylate. This reaction is convenientlyperformed in the presence of a base, for example, a tertiary amine suchas diisopropylethylamine. Convenient solvents include nitriles such asacetonitrile. The reaction is conveniently conducted at a temperature inthe range of from 0 to 100° C. The compounds of formula VI can beprepared by reacting a compound of formula IVa with a compound offormula XVIa′ or XVIa″:

Compounds of formula VI can also be prepared by reacting a compound offormula IVb with a compound of formula XVIb′ or XVIb″:

or a reactive derivative thereof such as an acid chloride or anhydride.These reactions are conveniently performed following, for example, themethod of process (b) described herein. Compounds of formula VII aregenerally known or can be prepared from readily available startingmaterials using well-known synthetic methods.

In process (d), the reducing agent may be, for example, hydrogen in thepresence of a Group VIII metal catalyst, such as palladium, or a metalhydride reducing agent such as a borohydride, including sodiumtriacetoxyborohydride. Convenient solvents include alcohols such asmethanol. The reaction is conveniently performed at a temperature in therange of from 0 to 100° C. The compounds of formula VIII may be preparedby oxidizing a compound corresponding to formula III in which L¹represents a hydroxyl group. Such oxidation reactions can be conducted,for example, using sulfur dioxide pyridine complex in dimethylsulfoxidein the presence of a tertiary amine such as diisopropylethylamine.

In process (e), the reducing agent may be, for example, hydrogen in thepresence of a Group VIII metal catalyst such as palladium, or a metalhydride reducing agent including borohydrides such as sodiumtriacetoxyborohydride, optionally used in combination with a titaniumtetraalkoxide such as titanium tetraisopropoxide. Convenient solventsinclude alcohols such as methanol, and halogenated hydrocarbons such asdichloromethane. The reaction is conveniently performed at a temperaturein the range of from 0 to 100° C. Compounds of formula IX may beprepared by reacting a compound of formula IVa with a compound offormula XVIIa′ or XVIIa″:

or a compound of formula IVb with a compound of formula XVIIb′ orXVIIb″:

in the presence of a carboxylic acid/amine coupling agent such as1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and1-hydroxybenzotriazole hydrate (HOBT) and the like.

As will be apparent to those skilled in the art, compounds of formula Iprepared by any of steps (a) to (e) herein may be further derivatized toform other compounds of formula I using methods and reagents well-knownin the art. By way of illustration, a compound of formula I may bereacted with bromine to afford a corresponding compound of formula I inwhich R², for example, represents a bromo group. Additionally, acompound of formula I in which R⁴ represents a hydrogen atom may bealkylated to afford a corresponding compound of formula I in which R⁴represents a (1-4C) alkyl group.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of the invention orintermediates thereof are described in the Examples set forth below.

Pharmaceutical Compositions and Formulations

The biphenyl compounds of the invention are typically administered to apatient in the form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to,inhaled, oral, nasal, topical (including transdermal) and parenteralmodes of administration.

It will be understood that any form of the compounds of the invention,(i.e., free base, pharmaceutically acceptable salt, solvate, etc.) thatis suitable for the particular mode of administration can be used in thepharmaceutical compositions discussed herein.

Accordingly, one embodiment of the invention is directed to apharmaceutical composition comprising a pharmaceutically acceptablecarrier or excipient and a therapeutically effective amount of acompound of formula I, or a pharmaceutically acceptable salt or solvateor stereoisomer thereof. The pharmaceutical composition may containother therapeutic and/or formulating agents if desired.

The pharmaceutical compositions of the invention typically contain atherapeutically effective amount of a compound of the invention or apharmaceutically acceptable salt or solvate or stereoisomer thereof, asthe active agent. Typically, such pharmaceutical compositions willcontain from about 0.01 to 95% by weight of the active agent; including,from about 0.01 to 30%, such as from about 0.01 to 10%.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combination of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the ingredients for such compositionsare commercially available from, for example, Sigma, P.O. Box 14508, St.Louis, Mo. 63178. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials that can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil, saffloweroil, sesame oil, olive oil, corn oil and soybean oil; glycols such aspropylene glycol; polyols such as glycerin, sorbitol, mannitol andpolyethylene glycol; esters such as ethyl oleate and ethyl laurate;agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; phosphate buffer solutions; compressedpropellant gases such as chlorofluorocarbons and hydrofluorocarbons; andother non-toxic compatible substances employed in pharmaceuticalcompositions.

The pharmaceutical compositions of the invention are typically preparedby thoroughly and intimately mixing or blending a compound of theinvention with a pharmaceutically acceptable carrier and one or moreoptional ingredients. If necessary or desired, the resulting uniformlyblended mixture can then be shaped or loaded into tablets, capsules,pills, canisters, cartridges, dispensers and the like using conventionalprocedures and equipment.

In one embodiment, the pharmaceutical compositions of the invention aresuitable for inhaled administration. Suitable pharmaceuticalcompositions for inhaled administration will typically be in the form ofan aerosol or a powder. Such compositions are generally administeredusing well-known delivery devices such as a nebulizer inhaler, ametered-dose inhaler (MDI), a dry powder inhaler (DPI) or a similardelivery device.

In a specific embodiment of the invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a nebulizer inhaler. Such nebulizer devices typically produce astream of high velocity air that causes the pharmaceutical compositioncomprising the active agent to spray as a mist that is carried into thepatient's respiratory tract. Accordingly, when formulated for use in anebulizer inhaler, the active agent is typically dissolved in a suitablecarrier to form a solution. Alternatively, the active agent can bemicronized and combined with a suitable carrier to form a suspension ofmicronized particles of respirable size, where micronized is typicallydefined as having about 90% or more of the particles with a diameter ofless than about 10 μm. Suitable nebulizer devices are commerciallyavailable, for example, by PARI GmbH (Stamberg, German). Other nebulizerdevices include Respimat (Boehringer Ingelheim) and those described, forexample, in U.S. Pat. No. 6,123,068 to Lloyd et al. and WO 97/12687(Eicher et al.), the disclosures of which are incorporated herein byreference in their entirety.

A representative pharmaceutical composition for use in a nebulizerinhaler comprises an isotonic aqueous solution comprising from about0.05 μg/mL to about 10 mg/mL of a compound of formula I or apharmaceutically acceptable salt or solvate or stereoisomer thereof.

In another specific embodiment of the invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a DPI. Such DPIs typically administer the active agent as afree-flowing powder that is dispersed in a patient's air-stream duringinspiration. In order to achieve a free flowing powder, the active agentis typically formulated with a suitable excipient such as lactose orstarch. Micronization is a common method of reducing crystal size tothat suitable for pulmonary delivery. Typically, the active agent ismicronized and combined with a suitable carrier to form a suspension ofmicronized particles of respirable size, where “micronized particles” or“micronized form” means at least about 90% of the particles have adiameter of less than about 10 μm. Other methods of reducing particlesize may also be used such as fine milling, chopping, crushing,grinding, milling, screening, trituration, pulverization, and so forth,as long as the desired particle size can be obtained.

A representative pharmaceutical composition for use in a DPI comprisesdry lactose having a particle size between about 1 μm and about 100 μmand micronized particles of a compound of formula I, or apharmaceutically acceptable salt or solvate or stereoisomer thereof.Such a dry powder formulation can be made, for example, by combining thelactose with the active agent and then dry blending the components.Alternatively, if desired, the active agent can be formulated without anexcipient. The pharmaceutical composition is then typically loaded intoa dry powder dispenser, or into inhalation cartridges or capsules foruse with a dry powder delivery device.

Examples of DPI delivery devices include Diskhaler (GlaxoSmithKline,Research Triangle Park, N.C.; see, e.g., U.S. Pat. No. 5,035,237 toNewell et al.); Diskus (GlaxoSmithKline; see, e.g., U.S. Pat. No.6,378,519 to Davies et al.); Turbuhaler (AstraZeneca, Wilmington, Del.;see, e.g., U.S. Pat. No. 4,524,769 to Wetterlin); Rotahaler(GlaxoSmithKline; see, e.g., U.S. Pat. No. 4,353,365 to Hallworth etal.) and Handihaler (Boehringer Ingelheim). Further examples of suitableDPI devices are described in U.S. Pat. No. 5,415,162 to Casper et al.,U.S. Pat. No. 5,239,993 to Evans, and U.S. Pat. No. 5,715,810 toArmstrong et al., and references cited therein. The disclosures of theaforementioned patents are incorporated herein by reference in theirentirety.

In yet another specific embodiment of the invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing an MDI, which typically discharges a measured amount of the activeagent or a pharmaceutically acceptable salt or solvate or stereoisomerthereof using compressed propellant gas. Accordingly, pharmaceuticalcompositions administered using an MDI typically comprise a solution orsuspension of the active agent in a liquefied propellant. Any suitableliquefied propellant may be employed including chlorofluorocarbons suchas CCl₃F, and hydrofluoroalkanes (HFAs) such as1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227). Due to concerns aboutchlorofluorocarbons affecting the ozone layer, formulations containingHFAs are generally preferred. Additional optional components of HFAformulations include co-solvents such as ethanol or pentane, andsurfactants such as sorbitan trioleate, oleic acid, lecithin, andglycerin. See, for example, U.S. Pat. No. 5,225,183 to Purewal et al.,EP 0717987 A2 (Minnesota Mining and Manufacturing Company), and WO92/22286 (Minnesota Mining and Manufacturing Company), the disclosuresof which are incorporated herein by reference in their entirety.

A representative pharmaceutical composition for use in a metered-doseinhaler comprises from about 0.01 to 5% by weight of a compound offormula I, or a pharmaceutically acceptable salt or solvate orstereoisomer thereof; from about 0 to 20% by weight ethanol; and fromabout 0 to 5% by weight surfactant; with the remainder being an HFApropellant.

Such compositions are typically prepared by adding chilled orpressurized hydrofluoroalkane to a suitable container containing theactive agent, ethanol (if present) and the surfactant (if present). Toprepare a suspension, the active agent is micronized and then combinedwith the propellant. The formulation is then loaded into an aerosolcanister, which forms a portion of a metered-dose inhaler device.Examples of metered-dose inhaler devices developed specifically for usewith HFA propellants are described in U.S. Pat. No. 6,006,745 to Mareckiand U.S. Pat. No. 6,143,277 to Ashurst et al. Alternatively, asuspension formulation can be prepared by spray drying a coating ofsurfactant on micronized particles of the active agent. See, forexample, WO 99/53901 (Glaxo Group Ltd.) and WO 00/61108 (Glaxo GroupLtd.). The disclosures of the aforementioned patents and publicationsare incorporated herein by reference in their entirety.

For additional examples of processes of preparing respirable particles,and formulations and devices suitable for inhalation dosing see U.S.Pat. No. 6,268,533 to Gao et al., U.S. Pat. No. 5,983,956 to Trofast;U.S. Pat. No. 5,874,063 to Briggner et al.; and U.S. Pat. No. 6,221,398to Jakupovic et al.; and WO 99/55319 (Glaxo Group Ltd.) and WO 00/30614(AstraZeneca AB); the disclosures of which are incorporated herein byreference in their entirety.

In another embodiment, the pharmaceutical compositions of the inventionare suitable for oral administration. Suitable pharmaceuticalcompositions for oral administration may be in the form of capsules,tablets, pills, lozenges, cachets, dragees, powders, granules; or as asolution or a suspension in an aqueous or non-aqueous liquid; or as anoil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup;and the like; each containing a predetermined amount of a compound ofthe invention as an active ingredient. The pharmaceutical compositionmay be packaged in a unit dosage form.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof the invention will typically comprise a compound of the presentinvention as the active ingredient and one or more pharmaceuticallyacceptable carriers such as sodium citrate or dicalcium phosphate.Optionally or alternatively, such solid dosage forms may also comprise:fillers or extenders such as starches, lactose, sucrose, glucose,mannitol, and/or silicic acid; binders such as carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia;humectants such as glycerol; disintegrating agents such as agar-agar,calcium carbonate, potato or tapioca starch, alginic acid, certainsilicates, and/or sodium carbonate; solution retarding agents such asparaffin; absorption accelerators such as quaternary ammonium compounds;wetting agents such as cetyl alcohol and/or glycerol monostearate;absorbents such as kaolin and/or bentonite clay; lubricants such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and/or mixtures thereof; coloring agents; andbuffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of the invention. Examples ofpharmaceutically acceptable antioxidants include: water-solubleantioxidants such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate sodium sulfite and the like; oil-solubleantioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA),butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and metal-chelating agents such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like. Coating agents for tablets,capsules, pills and like, include those used for enteric coatings suchas cellulose acetate phthalate (CAP), polyvinyl acetate phthalate(PVAP), hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate(CAT), carboxymethyl ethyl cellulose (CMEC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), and the like.

If desired, the pharmaceutical compositions of the invention may also beformulated to provide slow or controlled release of the activeingredient using, by way of example, hydroxypropyl methyl cellulose invarying proportions; or other polymer matrices, liposomes and/ormicrospheres.

In addition, the pharmaceutical compositions of the invention mayoptionally contain opacifying agents and may be formulated so that theyrelease the active ingredient only, or preferentially, in a certainportion of the gastrointestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Such liquid dosage formstypically comprise the active ingredient and an inert diluent such as,for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (e.g., cottonseed, groundnut, corn, germ,olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminium metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

The compounds of the invention can also be administered transdermallyusing known transdermal delivery systems and excipients. For example, acompound of the invention can be admixed with permeation enhancers suchas propylene glycol, polyethylene glycol monolaurate,azacycloalkan-2-ones and the like, and incorporated into a patch orsimilar delivery system. Additional excipients including gelling agents,emulsifiers and buffers, may be used in such transdermal compositions ifdesired.

The compounds of the invention can also be co-administered with othertherapeutic agents. This combination therapy involves using a compoundof the invention combined with one or more of these secondary agents,either formulated together (e.g., packaged together in a singleformulation) or formulated separately (e.g., packaged as separate unitdosage forms). Methods of formulating multiple agents together in thesame formulation or in separate unit dosage forms, are well known in theart.

The additional therapeutic agent(s) can be selected from otherbronchodilators (e.g., PDE₃ inhibitors, adenosine 2b modulators and β₂adrenergic receptor agonists); anti-inflammatory agents (e.g., steroidalanti-inflammatory agents such as corticosteroids; non-steroidalanti-inflammatory agents (NSAIDs), and PDE₄ inhibitors); othermuscarinic receptor antagonists (i.e., antichlolinergic agents);antiinfective agents (e.g., Gram positive and Gram negative antibioticsor antivirals); antihistamines; protease inhibitors; and afferentblockers (e.g., D₂ agonists and neurokinin modulators).

One particular embodiment of the invention is directed to a compositioncomprising (a) a pharmaceutically acceptable carrier and atherapeutically effective amount of a compound of formula I or apharmaceutically acceptable salt or solvate or stereoisomer thereof; and(b) a pharmaceutically acceptable carrier and a therapeuticallyeffective amount of an agent selected from a steroidal anti-inflammatoryagent such as a corticosteroid; a β₂ adrenergic receptor agonist; aphosphodiesterase-4 inhibitor; or a combination thereof; wherein thecompound of formula I and the agent are formulated together orseparately. In another embodiment, (b) is a pharmaceutically acceptablecarrier and a therapeutically effective amount of a β₂ adrenergicreceptor agonist and a steroidal anti-inflammatory agent. The secondaryagents can be used in the form of pharmaceutically acceptable salts orsolvates, and if appropriate, as optically pure stereoisomers.

Representative β₂ adrenergic receptor agonists that can be used incombination with compounds of the invention include, but are not limitedto, salmeterol, salbutamol, formoterol, salmefamol, fenoterol,terbutaline, albuterol, isoetharine, metaproterenol, bitolterol,pirbuterol, levalbuterol and the like, or pharmaceutically acceptablesalts thereof. Other β₂ adrenergic receptor agonists that can be usedinclude, but are not limited to,3-(4-{[6-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-phenyl]ethyl}amino)-hexyl]oxy}butyl)benzenesulfonamideand3-(-3-{[7-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}-amino)heptyl]oxy}-propyl)benzenesulfonamideand related compounds described in WO 02/066422 (Glaxo Group Ltd.);3-[3-(4-{[6-([(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino)hexyl]oxy}butyl)-phenyl]imidazolidine-2,4-dioneand related compounds described in WO 02/070490 (Glaxo Group Ltd.);3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)-hexyl]oxy}butyl)-benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamideand related compounds described in WO 02/076933 (Glaxo Group Ltd.);4-{(1R)-2-[(6-{2-[(2,6-dichlorobenzyl)oxy]ethoxy}hexyl)amino]-1-hydroxyethyl}-2-(hydroxymethyl)phenoland related compounds described in WO 03/024439 (Glaxo Group Ltd.);N-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamineand related compounds described in U.S. Pat. No. 6,576,793 to Moran etal.;N-{2-[4-(3-phenyl-4-methoxyphenyl)aminophenyl]ethyl}-(R)-2-hydroxy-2-(8-hydroxy-2(1H)-quinolinon-5-yl)ethylamineand related compounds described in U.S. Pat. No. 6,653,323 to Moran etal.; and pharmaceutically acceptable salts thereof. In a particularembodiment, the β₂-adrenoreceptor agonist is a crystallinemonohydrochloride salt of N-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamine.When employed, the β₂-adrenoreceptor agonist will be present in thepharmaceutical composition in a therapeutically effective amount.Typically, the β₂-adrenoreceptor agonist will be present in an amountsufficient to provide from about 0.05 μg to 500 μg per dose. Thedisclosures of the aforementioned patents and publications areincorporated herein by reference in their entirety.

Representative steroidal anti-inflammatory agents that can be used incombination with compounds of the invention include, but are not limitedto, methyl prednisolone, prednisolone, dexamethasone, fluticasonepropionate,6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester,6α,9═-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-androsta-1,4-diene-17β-carbothioicacid S-(2-oxo-tetrahydrofuran-3S-yl) ester, beclomethasone esters (e.g.,the 17-propionate ester or the 17,21-dipropionate ester), budesonide,flunisolide, mometasone esters (e.g., the furoate ester), triamcinoloneacetonide, rofleponide, ciclesonide, butixocort propionate, RPR-106541,ST-126 and the like, or pharmaceutically-acceptable salts thereof. Whenemployed, the steroidal anti-inflammatory agent will be present in thecomposition in a therapeutically effective amount. Typically, thesteroidal anti-inflammatory agent will be present in an amountsufficient to provide from about 0.05 μg to 500 μg per dose.

An exemplary combination is a compound of formula I, or pharmaceuticallyacceptable salt or solvate or stereoisomer thereof, co-administered withsalmeterol as the β₂ adrenergic receptor agonist, and fluticasonepropionate as the steroidal anti-inflammatory agent. Another exemplarycombination is a compound of formula I, or pharmaceutically acceptablesalt or solvate or stereoisomer thereof, co-administered with acrystalline monohydrochloride salt ofN-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamineas the β₂-adrenoreceptor agonist, and6α,9α-difluoro-17α-[(2-fluranylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester as the steroidal anti-inflammatory agent. Asnoted above, these agents can be formulated together or separately.

Other suitable combinations include, for example, otheranti-inflammatory agents, e.g., NSAIDs (e.g., sodium cromoglycate,nedocromil sodium, and phosphodiesterase (PDE) inhibitors such astheophylline, PDE4 inhibitors and mixed PDE3/PDE4 inhibitors);leukotriene antagonists (e.g., monteleukast); inhibitors of leukotrienesynthesis; iNOS inhibitors; protease inhibitors such as tryptase andelastase inhibitors; beta-2 integrin antagonists and adenosine receptoragonists or antagonists (e.g., adenosine 2a agonists); cytokineantagonists (e.g., chemokine antagonists such as, an interleukinantibody (αIL antibody), specifically, an αIL-4 therapy, an αIL-13therapy, or a combination thereof); or inhibitors of cytokine synthesis.

Representative phosphodiesterase-4 (PDE4) inhibitors or mixed PDE3/PDE4inhibitors that can be used in combination with the compounds of theinvention include, but are not limited to cis4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylicacid,2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-one;cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol];cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylicacid and the like, or pharmaceutically acceptable salts thereof. Otherrepresentative PDE4 or mixed PDE4/PDE3 inhibitors include AWD-12-281(elbion); NCS-613 (INSERM); D-4418 (Chiroscience and Schering-Plough);CI-1018 or PD-168787 (Pfizer); benzodioxole compounds described inWO99/16766 (Kyowa Hakko); K-34 (Kyowa Hakko); V-11294A (Napp);roflumilast (Byk-Gulden); pthalazinone compounds described in WO99/47505(Byk-Gulden); Pumafentrine (Byk-Gulden, now Altana); arofylline(Almirall-Prodesfarma); VM554/UM565 (Vemalis); T-440 (Tanabe Seiyaku);and T2585 (Tanabe Seiyaku).

Representative muscarinic antagonists (i.e., anticholinergic agents)that can be used in combination with the compounds of the inventioninclude, but are not limited to, atropine, atropine sulfate, atropineoxide, methylatropine nitrate, homatropine hydrobromide, hyoscyamine (d,l) hydrobromide, scopolamine hydrobromide, ipratropium bromide,oxitropium bromide, tiotropium bromide, methantheline, propanthelinebromide, anisotropine methyl bromide, clidinium bromide, copyrrolate(Robinul), isopropamide iodide, mepenzolate bromide, tridihexethylchloride (Pathilone), hexocyclium methylsulfate, cyclopentolatehydrochloride, tropicamide, trihexyphenidyl hydrochloride, pirenzepine,telenzepine, AF-DX 116 and methoctramine and the like, or apharmaceutically acceptable salt thereof; or, for those compounds listedas a salt, alternate pharmaceutically acceptable salt thereof.

Representative antihistamines (i.e., H₁-receptor antagonists) that canbe used in combination with the compounds of the invention include, butare not limited to, ethanolamines such as carbinoxamine maleate,clemastine fumarate, diphenylhydramine hydrochloride and dimenhydrinate;ethylenediamines such as pyrilamine amleate, tripelennaminehydrochloride and tripelennamine citrate; alkylamines such aschlorpheniramine and acrivastine; piperazines such as hydroxyzinehydrochloride, hydroxyzine pamoate, cyclizine hydrochloride, cyclizinelactate, meclizine hydrochloride and cetirizine hydrochloride;piperidines such as astemizole, levocabastine hydrochloride, loratadineor its descarboethoxy analogue, terfenadine and fexofenadinehydrochloride; azelastine hydrochloride; and the like, or apharmaceutically acceptable salt thereof; or, for those compounds listedas a salt, alternate pharmaceutically acceptable salt thereof.

Unless otherwise indicated, exemplary suitable doses for the othertherapeutic agents administered in combination with a compound of theinvention are in the range of about 0.05 μg/day to 100 mg/day.

The following formulations illustrate representative pharmaceuticalcompositions of the invention, as well as exemplary methods ofpreparation. One or more secondary agents can optionally be formulatedwith the compound of the invention (primary active agent). Alternately,the secondary agents(s) can be formulated separately and co-administeredwith the primary active agent, either simultaneously or sequentially.For example, in one embodiment, a single dry powder formulation can bemanufactured to include both the compound of the invention and one ormore secondary agents. In another embodiment, one formulation ismanufactured to contain the compound of the invention and separateformulation(s) are manufactured to contain the secondary agent(s). Suchdry powder formulations can then be packaged in separate blister packsand administered with a single DPI device.

Exemplary Dry Powder Formulation for Administration by Inhalation

0.2 mg of a compound of the invention is micronized and then blendedwith 25 mg of lactose. The blended mixture is then loaded into a gelatininhalation cartridge. The contents of the cartridge are administeredusing a powder inhaler.

Exemplary Dry Powder Formulation for Administration by a Dry PowderInhaler

A dry powder is prepared having a bulk formulation ratio of micronizedcompound of the invention (active agent) to lactose of 1:200. The powderis packed into a dry powder inhalation device capable of deliveringbetween about 10 μg and 100 μg of active agent per dose.

Exemplary Formulations for Administration by a Metered Dose Inhaler

A suspension containing 5 wt % of a compound of the invention (activeagent) and 0.1 wt % lecithin is prepared by dispersing 10 g of theactive agent as micronized particles with a mean size less than 10 μm ina solution formed from 0.2 g of lecithin dissolved in 200 mL ofdemineralized water. The suspension is spray dried and the resultingmaterial is micronized to particles having a mean diameter less than 1.5μm. The particles are loaded into cartridges with pressurized1,1,1,2-tetrafluoroethane.

Alternately, a suspension containing 5 wt % of the active agent, 0.5 wt% lecithin, and 0.5 wt % trehalose is prepared by dispersing 5 g of theactive agent as micronized particles with a mean size less than 10 μm ina colloidal solution formed from 0.5 g of trehalose and 0.5 g oflecithin dissolved in 100 mL of demineralized water. The suspension isspray dried and the resulting material is micronized to particles havinga mean diameter less than 1.5 μm. The particles are loaded intocanisters with pressurized 1,1,1,2-tetrafluoroethane.

Exemplary Aqueous Aerosol Formulation for Administration by Nebulizer

A pharmaceutical composition is prepared by dissolving 0.5 mg of acompound of the invention (active agent) in 1 mL of a 0.9% sodiumchloride solution acidified with citric acid. The mixture is stirred andsonicated until the active agent is dissolved. The pH of the solution isadjusted to a value in the range of from 3 to 8 (typically about 5) bythe slow addition of NaOH.

Exemplary Hard Gelatin Capsule Formulation for Oral Administration

The following ingredients are thoroughly blended and then loaded into ahard gelatin capsule: 250 mg of a compound of the invention, 200 mg oflactose (spray-dried), and 10 mg of magnesium stearate, for a total of460 mg of composition per capsule.

Exemplary Suspension Formulation for Oral Administration

The following ingredients are mixed to form a suspension containing 100mg of active ingredient per 10 mL of suspension.

Exemplary Injectable Formulation Ingredients Amount Compound of theinvention 1.0 g Fumaric acid 0.5 g Sodium chloride 2.0 g Methyl paraben0.15 g Propyl paraben 0.05 g Granulated sugar 25.5 g Sorbitol (70%solution) 12.85 g Veegum k (Vanderbilt Co.) 1.0 g Flavoring 0.035 mLColorings 0.5 mg Distilled water q.s. to 100 mL

The following ingredients are blended and the pH is adjusted to 4±0.5using 0.5 N HCl or 0.5 N NaOH.

UTILITY Ingredients Amount Compound of the invention 0.2 g Sodiumacetate buffer solution (0.4 M) 2.0 mL HCl (0.5 N) or NaOH (0.5 N) q.s.to pH 4 Water (distilled, sterile) q.s. to 20 mL

The biphenyl compounds of the invention are expected to be useful asmuscarinic receptor antagonists and therefore, such compounds areexpected to be useful for treating medical conditions mediated bymuscarinic receptors, i.e., medical conditions which are ameliorated bytreatment with a muscarinic receptor antagonist. Such medical conditionsinclude, by way of example, pulmonary disorders or diseases includingthose associated with reversible airway obstruction such as chronicobstructive pulmonary disease (e.g., chronic and wheezy bronchitis andemphysema), asthma, pulmonary fibrosis, allergic rhinitis, rhinorrhea,and the like. Other medical conditions that can be treated withmuscarinic receptor antagonists are genitourinary tract disorders suchas overactive bladder or detrusor hyperactivity and their symptoms;gastrointestinal tract disorders such as irritable bowel syndrome,diverticular disease, achalasia, gastrointestinal hypermotilitydisorders and diarrhea; cardiac arrhythmias such as sinus bradycardia;Parkinson's disease; cognitive disorders such as Alzheimer's disease;dismenorrhea; and the like.

In one embodiment, compounds of the invention are useful for treatingsmooth muscle disorders in mammals, including humans and their companionanimals (e.g., dogs, cats etc.). Such smooth muscle disorders include,by way of illustration, overactive bladder, chronic obstructivepulmonary disease and irritable bowel syndrome.

When used to treat smooth muscle disorders or other conditions mediatedby muscarinic receptors, compounds of the invention will typically beadministered orally, rectally, parenterally or by inhalation in a singledaily dose or in multiple doses per day. The amount of active agentadministered per dose or the total amount administered per day willtypically be determined by the patient's physician and will depend onsuch factors as the nature and severity of the patients condition, thecondition being treated, the age and general health of the patient, thetolerance of the patient to the active agent, the route ofadministration and the like.

Typically, suitable doses for treating smooth muscle disorders or otherdisorders mediated by muscarinic receptors will range from about 0.14μg/kg/day to 7 mg/kg/day of active agent; including from about 0.15μg/kg/day to 5 mg/kg/day. For an average 70 kg human, this would amountto about 10 μg to 500 mg per day of active agent.

In a specific embodiment, compounds of the invention are useful fortreating pulmonary or respiratory disorders, such as COPD or asthma, inmammals including humans. When used to treat such disorders, thecompounds of the invention will typically be administered by inhalationin multiple doses per day, in a single daily dose or a single weeklydose. Generally, the dose for treating a pulmonary disorder will rangefrom about 10 μg/day to 200 μg/day. As used herein, COPD includeschronic obstructive bronchitis and emphysema (see, for example, Bames,Chronic Obstructive Pulmonary Disease, N Engl J Med 343:269-78 (2000)).

When used to treat a pulmonary disorder, compounds of the invention areoptionally administered in combination with other therapeutic agentssuch as a β₂-adrenoreceptor agonist; a corticosteroid, a non-steroidalanti-inflammatory agent, or combinations thereof.

When administered by inhalation, compounds of the invention typicallyhave the effect of producing bronchodilation. Accordingly, oneembodiment of the invention is directed to a method of producingbronchodilation in a patient, comprising administering to a patient abronchodilation-producing amount of a compound of the invention.Generally, the therapeutically effective dose for producingbronchodilation will range from about 10 μg/day to 200 μg/day.

In another embodiment, the compounds of the invention are used to treatoveractive bladder. When used to treat overactive bladder, the compoundsof the invention will typically be administered orally in a single dailydose or in multiple doses per day; preferably in a single daily dose. Inone embodiment, the dose for treating overactive bladder will range fromabout 1.0 to 500 mg/day.

In yet another embodiment, compounds of the invention are used to treatirritable bowel syndrome. When used to treat irritable bowel syndrome,the compounds of the invention will typically be administered orally orrectally in a single daily dose or in multiple doses per day. In oneembodiment, the dose for treating irritable bowel syndrome will rangefrom about 1.0 to 500 mg/day.

Since compounds of the invention are muscarinic receptor antagonists,such compounds are also useful as research tools for investigating orstudying biological systems or samples having muscarinic receptors. Suchbiological systems or samples may comprise M₁, M₂, M₃, M₄ and/or M₅muscarinic receptors. Any suitable biological system or sample havingmuscarinic receptors may be employed in such studies, which may beconducted either in vitro or in vivo. Representative biological systemsor samples suitable for such studies include, but are not limited to,cells, cellular extracts, plasma membranes, tissue samples, mammals(such as mice, rats, guinea pigs, rabbits, dogs, pigs, etc.), and thelike.

In this embodiment, a biological system or sample comprising amuscarinic receptor is contacted with a muscarinic receptor-antagonizingamount of a compound of the invention. The effects of antagonizing themuscarinic receptor are then determined using conventional proceduresand equipment such as radioligand binding assays and functional assays.Such functional assays include ligand-mediated changes in intracellularcyclic adenosine monophosphate (cAMP), ligand-mediated changes inactivity of the enzyme adenylyl cyclase (which synthesizes cAMP),ligand-mediated changes in incorporation of guanosine5′-O-(γ-thio)triphosphate ([³⁵S]GTPγS) into isolated membranes viareceptor catalyzed exchange of [³⁵S]GTPγS for GDP, ligand-mediatedchanges in free intracellular calcium ions (measured, for example, witha fluorescence-linked imaging plate reader or FLIPR® from MolecularDevices, Inc.). Compounds of the invention will antagonize or decreasethe activation of muscarinic receptors in any of the functional assayslisted above, or assays of a similar nature. A muscarinicreceptor-antagonizing amount of a compound of the invention willtypically range from about 0.1 to 100 nanomolar.

Additionally, compounds of the invention can be used as research toolsfor discovering new compounds that have muscarinic receptor antagonistactivity. In this embodiment, muscarinic receptor binding data (e.g., asdetermined by in vitro radioligand displacement assays) for a testcompound or a group of test compounds is compared to the muscarinicreceptor binding data for a compound of the invention to identify thosetest compounds that have about equal or superior muscarinic receptorbinding, if any. This aspect of the invention includes, as separateembodiments, both the generation of comparison data (using theappropriate assays) and the analysis of the test data to identify testcompounds of interest.

In another embodiment, compounds of the invention are used to antagonizea muscarinic receptor in a biological system, and a mammal in particularsuch as mice, rats, guinea pigs, rabbits, dogs, pigs, humans and soforth. In this embodiment, a therapeutically effective amount of acompound of formula I is administered to the mammal. The effects ofantagonizing the muscarinic receptor can then determined usingconventional procedures and equipment, examples of which are describedabove.

Among other properties, compounds of the invention have been found to bepotent inhibitors of M₃ muscarinic receptor activity. Accordingly, in aspecific embodiment, the invention is directed to compounds of formula Ihaving an inhibition dissociation constant (K_(i)) for the M₃ receptorsubtype of less than or equal to 10 nM, as determined, for example, byan in vitro radioligand displacement assay. In one embodiment, compoundsof the invention have a K_(i) value for the M₃ receptor subtype of lessthan or equal to 5 nM.

Additionally, compounds of the invention are expected to possess adesirable duration of action. Accordingly, in another specificembodiment, the invention is directed to compounds of formula I having aduration of action greater than or equal to about 24 hours. Moreover,compounds of the invention are also expected to possess reduced sideeffects, such as dry mouth, at efficacious doses when administered byinhalation compared to other known muscarinic receptor antagonistsadministered by inhalation (such as tiotropium).

These and other properties, as well as the utility of the compounds, canbe demonstrated using various in vitro and in vivo assays that arewell-known to those skilled in the art. For example, representativeassays are described in further detail in the following Examples.

EXAMPLES

The Preparations and Examples illustrate specific embodiments of theinvention. The following abbreviations have the following meaningsunless otherwise indicated and any other abbreviations used herein andnot defined have their standard meaning:

AC adenylyl cyclase

ACh acetylcholine

ACN acetonitrile

BSA bovine serum albumin

cAMP 3′-5′ cyclic adenosine monophosphate

CHO Chinese hamster ovary

cM₅ cloned chimpanzee M₅ receptor

DCM dichloromethane (i.e., methylene chloride)

DIPEA N,N-diisopropylethylamine

DMF dimethylformamide

DMSO dimethyl sulfoxide

dPBS Dulbecco's phosphate buffered saline

EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

EDTA ethylenediaminetetraacetic acid

EtOAc ethyl acetate

EtOH ethanol

FBS fetal bovine serum

FLIPR fluorometric imaging plate reader

HATU O-(7-azabenzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate

HBSS Hank's buffered salt solution

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

hM₁ cloned human M₁ receptor

hM₂ cloned human M₂ receptor

hM₃ cloned human M₃ receptor

hM₄ cloned human M₄ receptor

hM₅ cloned human M₅ receptor

HOAc acetic acid

HOBT 1-hydroxybenzotriazole hydrate

IPA isopropanol

MCh methylcholine

MeOH methanol

Na(OAc)₃BH sodium triacetoxyborohydride

TFA trifluoroacetic acid

THF tetrahydrofuran

Unless noted otherwise, all materials, such as reagents, startingmaterials and solvents, were purchased from commercial suppliers (suchas Sigma-Aldrich, Fluka, and the like) and were used without furtherpurification.

Unless otherwise indicated, HPLC analysis was conducted using an Agilent(Palo Alto, Calif.) Series 1100 instrument equipped with a Zorbax BonusRP 2.1×50 mm column (Agilent) having a 3.5 micron particle size.Detection was by UV absorbance at 214 nm. The mobile phases employedwere as follows (by volume): A is ACN (2%), water (98%) and TFA (0.1%);and B is ACN (90%), water (10%) and TFA (0.1%). HPLC 10-70 data wasobtained using a flow rate of 0.5 mL/minute of 10 to 70% B over a 6minute gradient (with the remainder being A). Similarly, HPLC 5-35 dataand HPLC 10-90 data were obtained using 5 to 35% B; or 10 to 90% B overa 5 minute gradient.

Liquid chromatography mass spectrometry (LCMS) data were obtained withan Applied Biosystems (Foster City, Calif.) Model API-1 50EX instrument.LCMS 10-90 data was obtained using 10 to 90% Mobile Phase B over a 5minute gradient.

Small-scale purification was conducted using an API-150EX PrepWorkstation system from Applied Biosystems. The mobile phases employedwere as follows (by volume): A is water and 0.05% TFA; and B is ACN and0.05% TFA. For arrays (typically about 3 to 50 mg recovered sample size)the following conditions were used: 20 mL/min flow rate; 15 minutegradients and a 20 mm×50 mm Prism RP column with 5 micron particles(Thermo Hypersil-Keystone, Bellefonte, Pa.). For larger scalepurifications (typically greater than 100 mg crude sample), thefollowing conditions were used: 60 mL/min flow rate; 30 minute gradientsand a 41.4 mm×250 mm Microsorb BDS column with 10 micron particles(Varian, Palo Alto, Calif.).

Preparation 1 Biphenyl-2-ylcarbamic Acid Piperidin-4-yl Ester

Biphenyl-2-isocyanate (97.5 g, 521 mmol) and4-hydroxy-N-benzylpiperidine (105 g, 549 mmol) were heated together at70° C. for 12 hours. The reaction mixture was then cooled to 50° C. andEtOH (1 L) was added and then 6M HCl (191 mL) was added slowly. Theresulting mixture was then cooled to ambient temperature and ammoniumformate (98.5 g, 1.56 mol) was added and then nitrogen gas was bubbledthrough the solution vigorously for 20 minutes. Palladium on activatedcarbon (20 g, 10 wt % dry basis) was then added and the reaction mixturewas heated at 40° C. for 12 hours, and then filtered through a pad ofCelite. The solvent was then removed under reduced pressure and 1M HCl(40 mL) was added to the crude residue. The pH of the mixture was thenadjusted with 10 N NaOH to pH 12. The aqueous layer was extracted withEtOAc (2×150 mL) and the organic layer was dried (magnesium sulfate),filtered and the solvent removed under reduced pressure to give 155 g ofthe title intermediate (100% yield). HPLC (10-70) R_(t)=2.52; m/z:[M+H⁺] calcd for C₁₈H₂₀N₂O₂, 297.15; found, 297.3.

Preparation 2 3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionicAcid Methyl Ester

Methyl 3-bromopropionate (553 μL, 5.07 mmol) was added to a stirredsolution of biphenyl-2-ylcarbamic acid piperidin-4-yl ester (1.00 g,3.38 mmol; prepared as described in Preparation 1) and DIPEA (1.76 mL,10.1 mmol) in ACN (34 mL) at 50° C. and the reaction mixture was heatedat 50° C. overnight. The solvent was then removed under reducedpressure, and the residue was dissolved in DCM (30 mL). The resultingsolution was washed with saturated aqueous sodium bicarbonate solution(10 mL), dried (magnesium sulfate), filtered and the solvent was removedunder reduced pressure. The crude residue was purified by columnchromatography (5-10% MeOH/DCM) to give 905 mg of the title intermediate(70% yield).

Preparation 3 3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionicAcid

A stirred solution of3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionic acid methylester (902 mg, 2.37 mmol; prepared as described in Preparation 2) andlithium hydroxide (171 mg, 7.11 mmol) in 50% THF:H₂O (24 mL) was heatedat 30° C. overnight, and then acidified with concentrated HCl andlyophilized to give the title intermediate (˜100% yield, also containsLiCl salts).

Preparation 3A 3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionicAcid

A round bottomed flask was charged with biphenyl-2-ylcarbamic acidpiperidin-4-yl ester (50 g, 67.6 mmol, 1 equiv.; prepared as describedin Preparation 1) and 500 mL of DCM. Acrylic acid (15.05 mL, 100 mmol,1.3 equiv.) was added and the reaction was heated to 50° C. (reflux) for18 hours. The DCM was removed in vacuo and MeOH (600 mL) was added. TheMeOH solution was heated to 75° C. for 2 hours then allowed to cool toroom temperature, during which time a thick slurry formed. Theprecipitate was collected via vacuum filtration, washed with MeOH (50mL), and dried on the filter to afford 61 g of the title intermediate(98% yield).

Preparation 4 Biphenyl-2-ylcarbamic Acid1-[2-(5-aminopentylcarbamoyl)ethyl]piperidin-4-yl Ester

A mixture of 3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionicacid (3 g, 8.15 mmol; prepared as described in Preparation 3), HATU(4.65 g, 12.2 mmol), N-tert-butoxycarbonyl-1,5-diaminopentane (2.47 g,12.2 mmol) and DIPEA (4.25 mL, 24.52 mmol) in 100 mL of DCM was stirredat room temperature for 1 hour. The reaction mixture was washed with a1:1 solution of brine and 1N HCl (100 mL), water (100 mL), dried overmagnesium sulfate, filtered and concentrated. The residue was treatedwith 20% TFA in DCM (100 mL) at room temperature for 2 hours. Thesolvent and TFA were removed under reduced pressure. The residue wastaken up in 100 mL of DCM and washed with 1N NaOH (100 mL) and brine(100 mL). The organic layer was dried over magnesium sulfate, filteredand concentrated to give 4.36 g of the title compound as a solid.

Preparation 4A Biphenyl-2-ylcarbamic Acid1-[2-(5-aminopentylcarbamoyl)ethyl]piperidin-4-yl Ester

A mixture of 3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionicacid (98.6 g, 267 mmol, 1.0 equiv.; prepared as described in Preparation3A), diphenylphosphoryl azide (69 mL, 321 mmol, 1.2 equiv.),N-tert-butoxycarbonyl-1,5-diaminopentane (65.0 g, 321 mmol, 1.2 equiv.)and DIPEA (93 mL, 534 mmol, 2.0 equiv.) in DCM (790 mL) was stirred atroom temperature overnight. When the coupling was complete (determinedby HPLC), 4.0 M HCl in dioxane was added (267 mL, 1.068 mol, 4.0 equiv.)and the reaction was stirred overnight. The reaction was diluted withwater (1.0 L) and transferred to a separatory funnel. The DCM layer wasremoved and the aqueous layer was washed with isopropyl acetate (200mL). The pH of the aqueous layer was adjusted to 13-14 with solid NaOH,and the basic aqueous layer was extracted 3× isopropyl acetate (250 mL).Combined organic layers with washed with saturated brine (500 mL) anddried over anhydrous sodium sulfate. Solvent was removed to afford 126 gof the crude title compound, which was suitable for immediate use insubsequent synthetic steps, such as in Example 1.

Example 1 Biphenyl-2-ylcarbamic Acid1-(2-{5-[4-(3-dimethylaminopropoxy)benzylamino]pentylcarbamoyl}ethyl)piperidin-4-ylEster

Biphenyl-2-ylcarbamic acid1-[2-(5-aminopentylcarbamoyl)ethyl]piperidin-4-yl ester (45.2 mg, 0.1mmol; prepared as described in Preparation 4) was dissolved in 1 mL ofMeOH. To the solution was added[4-(3-dimethylaminopropoxy)phenyl]acetaldehyde (0.1 mmol) at roomtemperature. The reaction was stirred at room temperature for 30 minutesbefore it was treated with Na(OAc)₃BH (64 mg, 0.3 mmol). Stirring wascontinued for an additional 1 hour. The reaction mixture wasconcentrated, then dissolved in 1 mL of 1:1 HOAc/H₂O solution andpurified on reverse phase HPLC. The title compound was obtained as atri(trifluoroacetate) salt. MS m/z: [M+H⁺] calcd for C₃₈H₅₃N₅O₄, 644.42;found, 644.3.

The title compound can also be made in a similar manner usingbiphenyl-2-ylcarbamic acid1-[2-(5-aminopentylcarbamoyl)ethyl]piperidin-4-yl ester, prepared asdescribed in Preparation 4A.

Example 2

Following the procedure described in Example 1 and substituting theappropriate starting materials, reagents, and aldehyde, the followingcompounds were obtained as bis(trifluoroacetate) salts.

# Name p R⁷ R⁸ 2-1 Biphenyl-2-ylcarbamic acid 1-{2-[5-(4- 0 — —CH₃methoxybenzylamino)pentylcarbamoyl] ethyl}piperidin-4-yl ester. MS m/z:[M + H⁺] calcd for C₃₄H₄₄N₄O₄, 573.35; found, 573.3. 2-2Biphenyl-2-ylcarbamic acid 1-{2-[5-(3- 1 —O- —CH₃cyclopentyloxy-4-methoxybenzylamino) cyclopentylpentylcarbamoyl]-ethyl}piperidin-4-yl ester. MS m/z: [M + H⁺] calcd forC₃₉H₅₂N₄O₅, 657.40; found, 657.3. 2-3 Biphenyl-2-ylcarbamic acid1-{2-[5- 1 —OCH₃ —CH₃ (3,4-dimethoxybenzylamino)pentylcarbamoyl]ethyl}piperidin-4-yl ester. MS m/z: [M + H⁺] calcd forC₃₅H₄₆N₄O₅, 603.36; found, 603.2. 2-4 Biphenyl-2-ylcarbamic acid1-{2-[5-(4- 0 — —CF₃ trifluoromethoxybenzylamino)pentylcarbamoyl]ethyl}piperidin-4-yl ester. MS m/z: [M + H⁺] calcd for C₃₄H₄₁F₃N₄O₄,627.32; found, 627.3. 2-5 Biphenyl-2-ylcarbamic acid 1-{2-[5-(4- 0 ——CHF₂ difluoromethoxybenzylamino)pentylcarbamoyl]ethyl} piperidin-4-ylester. MS m/z: [M + H⁺] calcd for C₃₄H₄₂F₂N₄O₄, 609.33; found, 609.2.2-6 Biphenyl-2-ylcarbamic acid 1-{2-[5-(4- 0 — —C₆H₅phenoxybenzylamino)pentylcarbamoyl]- ethyl}piperidin-4-yl ester. MS m/z:[M + H⁺] calcd for C₃₉H₄₆N₄O₄, 635.36; found, 635.3.

Example 3

Following the procedure described in Example 1 and substituting theappropriate starting materials, reagents, and aldehyde, the followingcompounds were obtained as bis(trifluoroacetate) salts.

    #     Name

3-1 Biphenyl-2-ylcarbamic acid 1-(2-{5- [(benzo[1,3]dioxol-5-ylmethyl)amino]pentylcarbamoyl}ethyl) piperidin-4-yl ester. MS m/z: [M + H⁺]calcdfor C₃₄H₄₂N₄O₅, 587.33; found, 587.2.

3-2 Biphenyl-2-ylcarbamic acid 1-(2-{5- [(2,2-dimethylchroman-6-ylmethyl) amino]pentylcarbamoyl}ethyl) piperidin-4-yl ester.MS m/z: [M + H⁺]calcd for C₃₈H₅₀N₄O₄, 627.39; found, 627.3.

3-3 Biphenyl-2-ylcarbamic acid 1-(2-{5- [(2,3-dihydrobenzofuran-5-ylmethyl) amino]pentylcarbamoyl}ethyl) piperidin-4-ylester. MS m/z: [M + H⁺]calcd for C₃₅H₄₄N₄O₄, 585.35; found, 585.3.

3-4 Biphenyl-2-ylcarbamic acid 1-(2-{5-[(2,3-dihydro-benzo[1,4]dioxin-6- ylmethyl)amino]pentyl carbamoyl}ethyl)piperidin-4-yl ester. MS m/z: [M + H⁺]calcd for C₃₅H₄₄N₄O₅,601.34; found, 601.2.

Example 4 Biphenyl-2-ylcarbamic Acid 1-{2-[5-(4-Methoxybenzenesulfonylamino)pentylcarbamoyl]ethyl}piperidin-4-yl Ester

Biphenyl-2-ylcarbamic acid1-[2-(5-aminopentylcarbamoyl)ethyl]piperidin-4-yl ester (45.2 mg, 0.1mmol; prepared as described in Preparation 4) was dissolved in 0.5 mL ofACN. To the solution was added DIPEA (52 μL, 0.3 mmol) followed by4-methoxybenzenesulfonyl chloride (20.7 mg, 0.1 mmol) at roomtemperature. The reaction was stirred at room temperature for 2 hoursthen concentrated. The residue was dissolved in 1 mL of 1:1 HOAc/H₂Osolution and purified on reverse phase HPLC. The title compound wasobtained as a trifluoroacetate salt. MS m/z: [M+H⁺] calcd forC₃₃H₄₂N₄O₆S, 623.29; found, 623.2.

Preparation 5 Biphenyl-2-ylcarbamic Acid1-[2-(5-Oxopentylcarbamoyl)ethyl]piperidin-4-yl Ester

A mixture of 3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionicacid (5 g, 13.5 mmol; prepared as described in Preparation 3), HATU(10.3 g, 27 mmol), 5-amino-1-pentanol (1.67 g, 16.2 mmol) and DIPEA(7.04 mL, 40.5 mmol) in 100 mL of DCM was stirred at room temperaturefor 1 hour. The reaction mixture was then washed with brine (100 mL),water (100 mL), dried over magnesium sulfate, filtered and concentrated.The residue was dissolved in DCM (100 mL) and cooled down to −5° C. inan ice/brine bath. DIPEA (7.04 mL, 40.5 mmol) and DMSO (10 mL) wereadded to the solution, followed by sulfur trioxide pyridine complex(6.45 g, 40.5 mmol). The reaction mixture was stirred at 0° C. for 2hours and then washed with water (100 mL) and brine (2×100 mL). Theorganic layer was dried over magnesium sulfate, filtered andconcentrated to yield 4.88 g of the title intermediate (80% yield) as asemi-solid.

Example 5

Biphenyl-2-ylcarbamic acid1-[2-(5-oxopentylcarbamoyl)ethyl]piperidin-4-yl ester (45 mg, 0.1 mmol;prepared as described in Preparation 5) was dissolved in 1 mL of MeOH.To the solution was added 2-(4-methoxyphenyl)ethylamine (0.1 mmol) atroom temperature. The reaction was stirred at room temperature for 30minutes before it was treated with Na(OAc)₃BH (64 mg, 0.3 mmol).Stirring was continued for an additional 1 hour. The reaction mixturewas concentrated, then dissolved in 1 mL of 1:1 HOAc/H₂O solution andpurified on reverse phase HPLC. Compound 5-1 was obtained asbis(trifluoroacetate) salt.

Following the same procedure and substituting the appropriate amine,Compound 5-2 was obtained as a bis(trifluoroacetate) salt.

# Name X¹ R⁷ 5-1 Biphenyl-2-ylcarbamic acid 1-(2-{5-[2-(4-methoxyphenyl)—(CH₂)₂— —H ethylamino]pentylcarbamoyl}ethyl)piperidin- 4-yl ester. MSm/z: [M + H⁺] calcd for C₃₅H₄₆N₄O₄, 587.36; found, 587.3. 5-2Biphenyl-2-ylcarbamic acid 1-(2-{5-[2-(3,4- —(CH₂)₂— —OCH₃dimethoxyphenyl)ethylamino]pentylcarbamoyl}ethyl) piperidin-4-yl ester.MS m/z: [M + H⁺] calcd for C₃₆H₄₈N₄O₅, 617.37; found, 617.3.

Preparation 6 Biphenyl-2-ylcarbamic Acid1-[2-(4-Aminomethylphenylcarbamoyl)ethyl]piperidin-4-yl Ester

To a stirred solution of 4-(N-tert-butoxycarbonylaminomethyl)aniline(756 mg, 3.4 mmol),3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionic acid (1.5 g,4.08 mmol; prepared as described in Preparation 3) and HATU (1.55 g,4.08 mmol) in DMF (6.8 mL), was added DIPEA (770 μL, 4.42 mmol). Thereaction mixture was stirred at 50° C. overnight, and then the solventwas removed under reduced pressure. The resulting residue was dissolvedin DCM (20 mL) and washed with saturated aqueous sodium bicarbonatesolution (10 mL). The organic phase was then dried (magnesium sulfate)and the solvent was removed under reduced pressure. The crude productwas purified by flash chromatography (5-10% MeOH/DCM) to give a solid,which was dissolved in TFA/DCM (25%, 30 mL) and stirred at roomtemperature for 2 hours. The solvent was then removed under reducedpressure and the crude residue was dissolved in DCM (30 mL) and washedwith 1N NaOH (15 mL). The organic phase was separated, dried (magnesiumsulfate), filtered and the solvent was removed under reduced pressure togive 1.5 g of the title intermediate (94% yield over 2 steps).

Example 6

Biphenyl-2-ylcarbamic acid1-[2-(4-aminomethylphenylcarbamoyl)ethyl]piperidin-4-yl ester (47.2 mg,0.1 mmol; prepared as described in Preparation 6) was dissolved in 1 mLof MeOH. To the solution was added4-(3-dimethylaminopropoxy)benzaldehyde (0.1 mmol) at room temperature.The reaction was stirred at room temperature for 30 minutes before itwas treated with Na(OAc)₃BH (64 mg, 0.3 mmol). Stirring was continuedfor an additional 1 hour. The reaction mixture was concentrated, thendissolved in 1 mL of a 1:1 HOAc/H₂O solution and purified on reversephase HPLC. Compound 6-1 was obtained as a bis(trifluoroacetate) salt.

Compounds 6-2 to 6-6 were obtained by following the same procedure andsubstituting the appropriate aldehyde.

# Name p R⁷ R⁸ 6-1 Biphenyl-2-ylcarbamic acid 1-[2-(4- 0 ——(CH₂)₃N(CH₃)₂ {[4-(3-dimethylaminopropoxy)benzylamino]methyl}phenylcarbamoyl) ethyl]piperidin-4-yl ester. MS m/z:[M + H⁺] calcd for C₄₀H₄₉N₅O₄, 664.39; found, 664.3. 6-2Biphenyl-2-ylcarbamic acid 1-(2- 0 — —CF₃{4-[(4-trifluoromethoxybenzylamino) methyl]phenylcarbamoyl}ethyl)piperidin-4-yl ester. MS m/z: [M + H⁺] calcd for C₃₆H₃₇F₃N₄O₄,647.29; found, 647.2. 6-3 Biphenyl-2-ylcarbamic acid 1-(2- 0 — —CHF₂{4-[(4-difluoromethoxybenzylamino) methyl]phenylcarbamoyl}ethyl)piperidin-4-yl ester. MS m/z: [M + H⁺] calcd for C₃₆H₃₈F₂N₄O₄,629.30; found, 629.2. 6-4 Biphenyl-2-ylcarbamic acid 1-(2- 1 —O- —CH₃{4-[(3-cyclopentyloxy-4- cyclopentylmethoxybenzylamino)methyl]phenylcarbamoyl} ethyl)piperidin-4-yl ester.MS m/z: [M + H⁺] calcd for C₄₁H₄₈N₄O₅, 677.37; found, 677.3. 6-5Biphenyl-2-ylcarbamic acid 1-(2- 0 — —CH₃ {4-[(4-methoxybenzylamino)methyl]phenylcarbamoyl}ethyl) piperidin-4-yl ester. MS m/z: [M + H⁺]calcd for C₃₆H₄₀N₄O₄, 593.31; found, 593.2. 6-6 Biphenyl-2-ylcarbamicacid 1-[2-(4- taken together to form —O—CH₂—{[(benzo[1,3]dioxol-5-ylmethyl) amino]methyl}phenylcarbamoyl)ethyl]piperidin-4-yl ester. MS m/z: [M + H⁺] calcd for C₃₆H₃₈N₄O₅,607.29; found, 607.2.

Preparation 7 N-Benzyl-N-methylaminoacetaldehyde

To a 3-necked 2-L flask was added N-benzyl-N-methylethanolamine (30.5 g,0.182 mol), DCM (0.5 L), DIPEA (95 mL, 0.546 mol) and DMSO (41 mL, 0.728mol). Using an ice bath, the mixture was cooled to about −10° C. andsulfur trioxide pyridine-complex (87 g, 0.546 mol) was added in 4portions over 5 minute intervals. The reaction was stirred at −10° C.for 2 hours. Before removing the ice-bath, the reaction was quenched byadding water (0.5 L). The aqueous layer was separated and the organiclayer was washed with water (0.5 L) and brine (0.5 L) and then driedover magnesium sulfate and filtered to provide the title compound whichwas used without further purification.

Preparation 8 Biphenyl-2-ylcarbamic Acid1-[2-(Benzylmethylamino)ethyl]piperidin-4-yl Ester

To a 2-L flask, containing N-benzyl-N-methylaminoacetaldehyde (fromPreparation 7) in DCM (0.5 L) was added biphenyl-2-ylcarbamic acidpiperidin-4-yl ester (30 g, 0.101 mol; prepared as described as inPreparation 1) followed by Na(OAc)₃BH (45 g, 0.202 mol). The reactionmixture was stirred overnight and then quenched by the addition of 1 NHCl (0.5 L) with vigorous stirring. Three layers were observed and theaqueous layer was removed. After washing with 1N NaOH (0.5 L), ahomogenous organic layer was obtained, which was then washed with asaturated solution of aqueous NaCl (0.5 L), dried over magnesiumsulfate, filtered and the solvent removed under reduced pressure. Theresidue was purified by dissolving it in a minimal amount of isopropanoland cooling this solution to 0° C. to form a solid which was collectedand washed with cool isopropanol to provide 42.6 g of the title compound(95% yield). MS m/z: [M+H⁺] calcd for C₂₈H₃₃N₃O₂, 444.3; found, 444.6.R_(f)=3.51 min (10-70 ACN:H₂O, reverse phase HPLC).

Preparation 8A Biphenyl-2-ylcarbamic Acid1-[2-(Benzylmethylamino)ethyl]piperidin-4-yl Ester

The title compound was prepared by mesylation of N-benzyl-N-methylethanolamine, which was then reacted with biphenyl-2-ylcarbamic acidpiperidin-4-yl ester in an alkylation reaction.

A 500 mL flask (reactor flask) was charged withN-benzyl-N-methylethanolamine (24.5 mL), DCM (120 mL), NaOH (80 mL; 30wt %) and tetrabutylammonium chloride. Mixing at low speed throughoutthe reaction, the mixture was cooled to −10° C. (cooling bath), and theaddition funnel charged with DCM (30 mL) and mesyl chloride (15.85 mL),which was added drop wise at a constant rate over 30 minutes. Theaddition was exothermic, and stirring was continued for 15 minutes whilethe temperature equilibrated back to −10° C. The reaction was held forat least 10 minutes to ensure full hydrolysis of the excess mesylchloride.

A 250 mL flask was charged with biphenyl-2-ylcarbamic acidpiperidin-4-yl ester (26 g; prepared as described in Preparation 1) andDCM (125 mL), stirred for 15 minutes at room temperature, and themixture chilled briefly to 10° C. to form a slurry. The slurry was thencharged into the reactor flask via the addition funnel. The cooling bathwas removed and the reaction mixture was warmed to 5° C. The mixture wastransferred to a separatory funnel, the layers allowed to settle, andthe aqueous layer removed. The organic layer was transferred back to thereactor flask, stirring resumed, the mixture held to room temperature,and the reaction monitored by HPLC for a total of 3.5 hours.

The reactor flask was charged with NaOH (1M solution; 100 mL), stirred,and the layers allowed to settle. The organic layer was separated,washed (NaCl satd. solution), its volume partially reduced under vacuum,and subjected to repeated IPA washings. The solids were collected andallowed to air-dry (25.85 g, 98% purity). Additional solids wereobtained from further processing of the mother liquor (volume reduction,IPA, cooling).

Preparation 9 Biphenyl-2-ylcarbamic Acid1-(2-Methylaminoethyl)piperidin-4-yl Ester

To a Parr hydrogenation flask was added biphenyl-2-ylcarbamic acid1-[2-(benzylmethylamino)ethyl]piperidin-4-yl ester (40 g, 0.09 mol;prepared as described in Preparation 8) and EtOH (0.5 L). The flask wasflushed with nitrogen gas and palladium on activated carbon (15 g, 10 wt% (dry basis), 37% wt/wt) was added along with HOAc (20 mL). The mixturewas kept on the Parr hydrogenator under a hydrogen atmosphere (˜50 psi)for 3 hours. The mixture was then filtered and washed with EtOH. Thefiltrate was condensed and the residue was dissolved in a minimal amountof DCM. Isopropyl acetate (10 volumes) was added slowly to form a solidwhich was collected to provide 22.0 g of the title compound (70% yield).MS m/z: [M+H⁺] calcd for C₂₁H₂₇N₃O₂, 354.2; found, 354.3. R_(f)=2.96 min(10-70 ACN:H₂O, reverse phase HPLC).

Preparation 10 Biphenyl-2-ylcarbamic Acid1-{2-[(4-Formylbenzoyl)methylamino]ethyl}piperidin-4-yl Ester

To a three-necked 1-L flask was added 4-carboxybenzaldehyde (4.77 g,31.8 mmol), EDC (6.64 g, 34.7 mmol), HOBT (1.91 g, 31.8 mmol), and DCM(200 mL). When the mixture was homogenous, a solution ofbiphenyl-2-ylcarbamic acid 1-(2-methylaminoethyl)piperidin-4-yl ester(10 g, 31.8 mmol; prepared as described in Preparation 9) in DCM (100mL) was added slowly. The reaction mixture was stirred at roomtemperature for 16 hours and then washed with water (1×100 mL), 1N HCl(5×60 mL), 1N NaOH (1×100 mL) brine (1×50 mL), dried over sodiumsulfate, filtered and concentrated to afford 12.6 g of the titlecompound (92% yield; 85% purity based on HPLC). MS m/z: [M+H⁺] calcd forC₂₉H₃₁N₃O₄, 486.2; found, 486.4. R_(f)=3.12 min (10-70 ACN:H₂O, reversephase HPLC).

Example 7

To a stirred solution of biphenyl-2-ylcarbamic acid1-{2-[(4-formylbenzoyl)methylamino]ethyl}piperidin-4-yl ester (48 mg,0.1 mmol; prepared as described in Preparation 10) in a 1:1 solution ofDCM/MeOH (1 mL) was added 4-methoxybenzylaldehyde (16 mg, 0.12 mmol),then sodium triacetoxyborohydride (27 mg, 0.12 mmol). The mixture wasstirred for 2 hours and then the solvent was removed under reducedpressure. A 1:1 HOAc/H₂O solution (1.5 mL) was added to the reactionmixture. The mixture was chromatographed on reverse-phase HPLC (gradientelution, 10-50% ACN/H₂O) to afford 3.1 mg of Compound 7-1 as abis(trifluoroacetate) salt.

Compound 7-2 was synthesized in a similar manner.

    #     Name

7-1 Biphenyl-2-ylcarbamic acid 1-[2-({4- [(4-methoxybenzylamino)methyl]benzoyl}methyl amino)ethyl]piperidin- 4-yl ester. MS m/z: [M + H⁺]calcdfor C₃₇H₄₂N₄O₄, 607.33; found, 607.3.

7-2 Biphenyl-2-ylcarbamic acid 1-[2-({4- [(2-methoxybenzylamino)methyl]benzoyl}methyl amino)ethyl]piperidin- 4-yl ester. MS m/z: [M + H⁺]calcdfor C₃₇H₄₂N₄O₄, 607.33; found, 607.3.

Assay 1 Radioligand Binding Assay Membrane Preparation from CellsExpressing hM₁, hM₂, hM₃ and hM₄ Muscarinic Receptor Subtypes

CHO cell lines stably expressing cloned human hM₁, hM₂, hM₃ and hM₄muscarinic receptor subtypes, respectively, were grown to nearconfluency in medium consisting of HAM's F-12 supplemented with 10% FBSand 250 μg/mL Geneticin. The cells were grown in a 5% CO₂, 37° C.incubator and lifted with 2 mM EDTA in dPBS. Cells were collected by 5minute centrifugation at 650×g, and cell pellets were either storedfrozen at −80° C. or membranes were prepared immediately. For membranepreparation, cell pellets were resuspended in lysis buffer andhomogenized with a Polytron PT-2100 tissue disrupter (Kinematica AG; 20seconds×2 bursts). Crude membranes were centrifuged at 40,000×g for 15minutes at 4° C. The membrane pellet was then resuspended withresuspension buffer and homogenized again with the Polytron tissuedisrupter. The protein concentration of the membrane suspension wasdetermined by the method described in Lowry, O. et al., Journal ofBiochemistry 193:265 (1951). All membranes were stored frozen inaliquots at −80° C. or used immediately. Aliquots of prepared hM₅receptor membranes were purchased directly from Perkin Elmer and storedat −80° C. until use.

Radioligand Binding Assay on Muscarinic Receptor Subtypes hM₁, hM₂, hM₃,hM₄ and hM₅

Radioligand binding assays were performed in 96-well microtiter platesin a total assay volume of 100 μL. CHO cell membranes stably expressingeither the hM₁, hM₂, hM₃, hM₄ or hM₅ muscarinic subtype were diluted inassay buffer to the following specific target protein concentrations(μg/well): 10 μg for hM₁, 10-15 μg for hM₂, 10-20 μg for hM₃, 10-20 μgfor hM₄, and 10-12 μg for hM₅. The membranes were briefly homogenizedusing a Polytron tissue disruptor (10 seconds) prior to assay plateaddition. Saturation binding studies for determining K_(D) values of theradioligand were performed using L-[N-methyl-³H]scopolamine methylchloride ([³H]-NMS) (TRK666, 84.0 Ci/mmol, Amersham Pharmacia Biotech,Buckinghamshire, England) at concentrations ranging from 0.001 nM to 20nM. Displacement assays for determination of K_(i) values of testcompounds were performed with [³H]-NMS at 1 nM and eleven different testcompound concentrations. The test compounds were initially dissolved toa concentration of 400 μM in dilution buffer and then serially diluted5× with dilution buffer to final concentrations ranging from 10 pM to100 μM. The addition order and volumes to the assay plates were asfollows: 25 μL radioligand, 25 μL diluted test compound, and 50 μLmembranes. Assay plates were incubated for 60 minutes at 37° C. Bindingreactions were terminated by rapid filtration over GF/B glass fiberfilter plates (PerkinElmer Inc., Wellesley, Mass.) pre-treated in 1%BSA. Filter plates were rinsed three times with wash buffer (10 mMHEPES) to remove unbound radioactivity. Plates were then air dried, and50 μL Microscint-20 liquid scintillation fluid (PerkinElmer Inc.,Wellesley, Mass.) was added to each well. The plates were then countedin a PerkinElmer Topcount liquid scintillation counter (PerkinElmerInc., Wellesley, Mass.). Binding data were analyzed by nonlinearregression analysis with the GraphPad Prism Software package (GraphPadSoftware, Inc., San Diego, Calif.) using the one-site competition model.K_(i) values for test compounds were calculated from observed IC₅₀values and the K_(D) value of the radioligand using the Cheng-Prusoffequation (Cheng Y; Prusoff W. H. Biochemical Pharmacology22(23):3099-108 (1973)). K_(i) values were converted to pK_(i) values todetermine the geometric mean and 95% confidence intervals. These summarystatistics were then converted back to K_(i) values for data reporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher binding affinity for the receptor tested. Exemplary compoundsof the invention that were tested in this or a similar assay, typicallywere found to have a K_(i) value of less than about 10 nM for the M₃muscarinic receptor subtype, with many compounds having a K_(i) value ofless than about 5 nM.

Assay 2 Muscarinic Receptor Functional Potency Assays Blockade ofAgonist-Mediated Inhibition of cAMP Accumulation

In this assay, the functional potency of a test compound is determinedby measuring the ability of the test compound to blockoxotremorine-inhibition of forskolin-mediated cAMP accumulation inCHO-K1 cells expressing the hM₂ receptor. cAMP assays are performed in aradioimmunoassay format using the Flashplate Adenylyl Cyclase ActivationAssay System with ¹²⁵I-cAMP (NEN SMP004B, PerkinElmer Life SciencesInc., Boston, Mass.), according to the manufacturer's instructions.

Cells are rinsed once with dPBS and lifted with Trypsin-EDTA solution(0.05% trypsin/0.53 mM EDTA) as described in the Cell Culture andMembrane Preparation section above. The detached cells are washed twiceby centrifugation at 650×g for five minutes in 50 mLs dPBS. The cellpellet is then re-suspended in 10 mL dPBS, and the cells are countedwith a Coulter Z1 Dual Particle Counter (Beckman Coulter, Fullerton,Calif.). The cells are centrifuged again at 650×g for five minutes andre-suspended in stimulation buffer to an assay concentration of1.6×10⁶−2.8×10⁶ cells/mL. The test compound is initially dissolved to aconcentration of 400 μM in dilution buffer (dPBS supplemented with 1mg/mL BSA (0.1%)), and then serially diluted with dilution buffer tofinal molar concentrations ranging from 100 μM to 0.1 nM. Oxotremorineis diluted in a similar manner.

To measure oxotremorine inhibition of adenylyl cyclase (AC) activity, 25μL forskolin (25 μM final concentration diluted in dPBS), 25 μL dilutedoxotremorine, and 50 μL cells are added to agonist assay wells. Tomeasure the ability of a test compound to block oxotremorine-inhibitedAC activity, 25 μL forskolin and oxotremorine (25 μM and 5 μM finalconcentrations, respectively, diluted in dPBS) 25 μL diluted testcompound, and 50 μL cells are added to remaining assay wells. Reactionsare incubated for 10 minutes at 37° C. and stopped by addition of 100 μLice-cold detection buffer. Plates are sealed, incubated overnight atroom temperature and counted the next morning on a PerkinElmer TopCountliquid scintillation counter (PerkinElmer Inc., Wellesley, Mass.). Theamount of cAMP produced (pmol/well) is calculated based on the countsobserved for the samples and cAMP standards, as described in themanufacturer's user manual. Data are analyzed by nonlinear regressionanalysis with the GraphPad Prism Software package (GraphPad Software,Inc., San Diego, Calif.) using the non-linear regression, one-sitecompetition equation. The Cheng-Prusoff equation is used to calculatethe K_(i), using the EC₅₀ of the oxotremorine concentration-responsecurve and the oxotremorine assay concentration as the K_(D) and [L],respectively. The K_(i) values are converted to pK_(i) values todetermine the geometric mean and 95% confidence intervals. These summarystatistics are then converted back to K_(i) values for data reporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. Compounds of theinvention are expected to have a K_(i) value of less than about 10 nMfor blockade of oxotremorine-inhibition of forskolin-mediated cAMPaccumulation in CHO-K1 cells expressing the hM₂ receptor, when tested inthis or a similar assay.

Blockade of Agonist-Mediated [³S]GTPγS Binding

In a second functional assay, the functional potency of test compoundscan be determined by measuring the ability of the compounds to blockoxotremorine-stimulated [³⁵S]GTPγS binding in CHO-K1 cells expressingthe hM₂ receptor.

At the time of use, frozen membranes are thawed and then diluted inassay buffer with a final target tissue concentration of 5-10 μg proteinper well. The membranes are briefly homogenized using a Polytron PT-2100tissue disrupter and then added to the assay plates. The EC₉₀ value(effective concentration for 90% maximal response) for stimulation of[³⁵S]GTPγS binding by the agonist oxotremorine is determined in eachexperiment.

To determine the ability of a test compound to inhibitoxotremorine-stimulated [³⁵S]GTPγS binding, the following is added toeach well of 96 well plates: 25 μL of assay buffer with [³⁵S]GTPγS (0.4nM), 25 μL of oxotremorine(EC₉₀) and GDP (3 μM), 25 μL of diluted testcompound and 25 μL CHO cell membranes expressing the hM₂ receptor. Theassay plates are then incubated at 37° C. for 60 minutes. The assayplates are filtered over. 1% BSA-pretreated GF/B filters using aPerkinElmer 96-well harvester. The plates are rinsed with ice-cold washbuffer for 3×3 seconds and then air or vacuum dried. Microscint-20scintillation liquid (50 μL) is added to each well, and each plate issealed and radioactivity counted on a topcounter (PerkinElmer). Data areanalyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using thenon-linear regression, one-site competition equation. The Cheng-Prusoffequation is used to calculate the K_(i), using the IC₅₀ values of theconcentration-response curve for the test compound and the oxotremorineconcentration in the assay as the K_(D) and [L], ligand concentration,respectively.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. Compounds of theinvention are expected to have a K_(i) value of less than about 10 nMfor blockade of oxotremorine-stimulated [³⁵S]GTPγS binding in CHO-K1cells expressing the hM₂ receptor, when tested in this or a similarassay.

Blockade of Agonist-Mediated Calcium Release via FLIPR Assays

Muscarinic receptor subtypes (M₁, M₃ and M₅ receptors), which couple toG_(q) proteins, activate the phospholipase C (PLC) pathway upon agonistbinding to the receptor. As a result, activated PLC hydrolyzesphosphatyl inositol diphosphate (PIP₂) to diacylglycerol (DAG) andphosphatidyl-1,4,5-triphosphate (IP₃), which in turn generates calciumrelease from intracellular stores, i.e., endoplasmic and sarcoplasmicreticulum. The FLIPR (Molecular Devices, Sunnyvale, Calif.) assaycapitalizes on this increase in intracellular calcium by using a calciumsensitive dye (Fluo-4AM, Molecular Probes, Eugene, Oreg.) thatfluoresces when free calcium binds. This fluorescence event is measuredin real time by the FLIPR, which detects the change in fluorescence froma monolayer of cells cloned with human M₁ and M₃, and chimpanzee M₅receptors. Antagonist potency can be determined by the ability ofantagonists to inhibit agonist-mediated increases in intracellularcalcium.

For FLIPR calcium stimulation assays, CHO cells stably expressing thehM₁, hM₃ and cM₅ receptors are seeded into 96-well FLIPR plates thenight before the assay is done. Seeded cells are washed twice byCellwash (MTX Labsystems, Inc.) with FLIPR buffer (10 mM HEPES, pH 7.4,2 mM calcium chloride, 2.5 mM probenecid in Hank's Buffered SaltSolution (HBSS) without calcium and magnesium) to remove growth mediaand leaving 50 μL/well of FLIPR buffer. The cells are then incubatedwith 50 μL/well of 4 μM FLUO-4AM (a 2× solution was made) for 40 minutesat 37° C., 5% carbon dioxide. Following the dye incubation period, cellsare washed two times with FLIPR buffer, leaving a final volume of 50μL/well.

To determine antagonist potency, the dose-dependent stimulation ofintracellular Ca²⁺ release for oxotremorine is first determined so thatantagonist potency can later be measured against oxotremorinestimulation at an EC₉₀ concentration. Cells are first incubated withcompound dilution buffer for 20 minutes, followed by agonist addition,which is performed by the FLIPR. An EC₉₀ value for oxotremorine isgenerated according to the method detailed in the FLIPR measurement anddata reduction section below, in conjunction with the formulaEC_(F)=((F/100−F)^1/H)*EC₅₀. An oxotremorine concentration of 3×EC_(F)is prepared in stimulation plates such that an EC₉₀ concentration ofoxotremorine is added to each well in the antagonist inhibition assayplates.

The parameters used for the FLIPR are: exposure length of 0.4 seconds,laser strength of 0.5 watts, excitation wavelength of 488 nm, andemission wavelength of 550 nm. Baseline is determined by measuring thechange in fluorescence for 10 seconds prior to addition of agonist.Following agonist stimulation, the FLIPR continuously measured thechange of fluorescence every 0.5 to 1 second for 1.5 minutes to capturethe maximum fluorescence change. The change of fluorescence is expressedas maximum fluorescence minus baseline fluorescence for each well. Theraw data is analyzed against the logarithm of drug concentration bynonlinear regression with GraphPad Prism (GraphPad Software, Inc., SanDiego, Calif.) using the built-in model for sigmoidal dose-response.Antagonist K_(i) values are determined by Prism using the oxotremorineEC₅₀ value as the K_(D) and the oxotremorine EC₉₀ for the ligandconcentration according to the Cheng-Prusoff equation (Cheng & Prusoff,1973).

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. Compounds of theinvention are expected to have a K_(i) value of less than about 10 nMfor blockade of agonist-mediated calcium release in CHO cells stablyexpressing the hM₃ receptor, when tested in this or a similar assay.

Assay 3 Determination of Duration of Bronchoprotection in Guinea PigModel of Acetylcholine-Induced Bronchoconstriction

This in vivo assay is used to assess the bronchoprotective effects oftest compounds exhibiting muscarinic receptor antagonist activity.Groups of six male guinea pigs (Duncan-Hartley (HsdPoc:DH) Harlan,Madison, Wis.) weighing between 250 and 350 g are individuallyidentified by cage cards. Throughout the study animals are allowedaccess to food and water ad libitum.

Test compounds are administered via inhalation over 10 minutes in awhole-body exposure dosing chamber (R&S Molds, San Carlos, Calif.). Thedosing chambers are arranged so that an aerosol was simultaneouslydelivered to 6 individual chambers from a central manifold. Guinea pigsare exposed to an aerosol of a test compound or vehicle (WFI). Theseaerosols are generated from aqueous solutions using an LC Star NebulizerSet (Model 22F51, PARI Respiratory Equipment, Inc. Midlothian, Va.)driven by a mixture of gases (CO₂=5%, O₂=21% and N₂=74%) at a pressureof 22 psi. The gas flow through the nebulizer at this operating pressureis approximately 3 L/minute. The generated aerosols are driven into thechambers by positive pressure. No dilution air is used during thedelivery of aerosolized solutions. During the 10 minute nebulization,approximately 1.8 mL of solution is nebulized. This is measuredgravimetrically by comparing pre-and post-nebulization weights of thefilled nebulizer.

The bronchoprotective effects of test compounds administered viainhalation are evaluated using whole body plethysmography at 1.5, 24, 48and 72 hours post-dose. Forty-five minutes prior to the start of thepulmonary evaluation, each guinea pig is anesthetized with anintramuscular injection of ketamine (43.75 mg/kg), xylazine (3.50 mg/kg)and acepromazine (1.05 mg/kg). After the surgical site is shaved andcleaned with 70% alcohol, a 2-3 cm midline incision of the ventralaspect of the neck was made. Then, the jugular vein is isolated andcannulated with a saline-filled polyethylene catheter (PE-50, BectonDickinson, Sparks, Md.) to allow for intravenous infusions of ACh(Sigma-Aldrich, St. Louis, Mo.) in saline. The trachea is then dissectedfree and cannulated with a 14G teflon tube (#NE-014, Small Parts, MiamiLakes, Fla.). If required, anesthesia is maintained by additionalintramuscular injections of the aforementioned anesthetic mixture. Thedepth of anesthesia is monitored and adjusted if the animal responds topinching of its paw or if the respiration rate is greater than 100breaths/minute.

Once the cannulations are complete, the animal is placed into aplethysmograph (#PLY3114, Buxco Electronics, Inc., Sharon, Conn.) and anesophageal pressure cannula (PE-160, Becton Dickinson, Sparks, Md.) isinserted to measure pulmonary driving pressure (pressure). The teflontracheal tube is attached to the opening of the plethysmograph to allowthe guinea pig to breathe room air from outside the chamber. The chamberis then sealed. A heating lamp is used to maintain body temperature andthe guinea pig's lungs are inflated 3 times with 4 mL of air using a 10mL calibration syringe (#5520 Series, Hans Rudolph, Kansas City, Mo.) toensure that the lower airways do not collapse and that the animal doesnot suffer from hyperventilation.

Once it is determined that baseline values are within the range 0.3-0.9mL/cm H₂O for compliance and within the range 0.1-0.199 cm H₂O/mL persecond for resistance, the pulmonary evaluation is initiated. A Buxcopulmonary measurement computer progam enables the collection andderivation of pulmonary values. Starting this program initiates theexperimental protocol and data collection. The changes in volume overtime that occur within the plethysmograph with each breath are measuredvia a Buxco pressure transducer. By integrating this signal over time, ameasurement of flow is calculated for each breath. This signal, togetherwith the pulmonary driving pressure changes, which are collected using aSensym pressure transducer (#TRD4100), is connected via a Buxco (MAX2270) preamplifier to a data collection interface (#'s SFT3400 andSFT3813). All other pulmonary parameters are derived from these twoinputs.

Baseline values are collected for 5 minutes, after which time the guineapigs are challenged with ACh. ACh (0.1 mg/mL) is infused intravenouslyfor 1 minute from a syringe pump (sp210iw, World Precision Instruments,Inc., Sarasota, Fla.) at the following doses and prescribed times fromthe start of the experiment: 1.9 μg/minute at 5 minutes, 3.8 μg/minuteat 10 minutes, 7.5 μg/minute at 15 minutes, 15.0 μg/minute at 20minutes, 30 μg/minute at 25 minutes and 60 μg/minute at 30 minutes. Ifresistance or compliance has not returned to baseline values at 3minutes following each ACh dose, the guinea pig's lungs are inflated 3times with 4 mL of air from a 10 mL calibration syringe. Recordedpulmonary parameters includes respiration frequency (breaths/minute),compliance (mL/cm H₂O) and pulmonary resistance (cm H₂O/mL per second).Once the pulmonary function measurements are completed at minute 35 ofthis protocol, the guinea pig is removed from the plethysmograph andeuthanized by carbon dioxide asphyxiation.

The data are evaluated in one or both of the following ways:

(a) Pulmonary resistance (R_(L), cm H₂O/mL per second) is calculatedfrom the ratio of “change in pressure” to “the change in flow.” TheR_(L) response to ACh (60 μg/min, IH) is computed for the vehicle andthe test compound groups. The mean ACh response in vehicle-treatedanimals, at each pre-treatment time, is calculated and used to compute %inhibition of ACh response, at the corresponding pre-treatment time, ateach test compound dose. Inhibition dose-response curves for ‘R_(L)’ arefitted with a four parameter logistic equation using GraphPad Prism,version 3.00 for Windows (GraphPad Software, San Diego, Calif.) toestimate bronchoprotective ID₅₀ (dose required to inhibit the ACh (60μg/min) bronchoconstrictor response by 50%). The equation used is asfollows:Y=Min+(Max−Min)/(1+10^(((log ID50-X)*Hillslope)))where X is the logarithm of dose, Y is the response (% Inhibition of AChinduced increase in R_(L)). Y starts at Min and approachesasymptotically to Max with a sigmoidal shape.

(b) The quantity PD₂, which is defined as the amount of ACh or histamineneeded to cause a doubling of the baseline pulmonary resistance, iscalculated using the pulmonary resistance values derived from the flowand the pressure over a range of ACh or histamine challenges using thefollowing equation (which is derived from a equation used to calculatePC₂₀ values described in American Thoracic Society. Guidelines formethacholine and exercise challenge testing—1999. Am J Respir Crit CareMed. 161:309-329 (2000)):

${PD}_{2} = {{antilog}\mspace{11mu}\lbrack {{\log\; C_{1}} + \frac{( {{\log\mspace{11mu} C_{2}} - {\log\mspace{11mu} C_{1}}} )( {{2R_{0}} - R_{1}} )}{R_{2} - R_{1}}} \rbrack}$where: C₁ is the concentration of ACh or histamine preceding C₂; C₂ isthe concentration of ACh or histamine resulting in at least a 2-foldincrease in pulmonary resistance (R_(L)); R₀ is the baseline R_(L)value; R₁ is the R_(L) value after C₁; and R₂ is the R_(L) value afterC₂. An efficacious dose is defined as a dose that limits thebronchrestriction response to a 50 mg/mL dose of ACh to a doubling ofthe baseline pulmonary resistance (PD₂₍₅₀₎).

Statistical analysis of the data is performed using a two-tailedStudents t-test. A P-value <0.05 is considered significant. Generally,test compounds having a PD₂₍₅₀₎ less than about 200 μg/mL forACh-induced bronchoconstriction at 1.5 hours post-dose in this assay arepreferred. Compounds of the invention are expected to have a PD₂₍₅₀₎ ofless than about 200 μg/mL for ACh-induced bronchoconstriction at 1.5hours post-dose, when tested in this or a similar assay.

Assay 4 Inhalation Guinea Pig Salivation Assay

Guinea pigs (Charles River, Wilmington, Mass.) weighing 200-350 g areacclimated to the in-house guinea pig colony for at least 3 daysfollowing arrival. Test compound or vehicle are dosed via inhalation(IH) over a 10 minute time period in a pie shaped dosing chamber (R&SMolds, San Carlos, Calif.). Test solutions are dissolved in sterilewater and delivered using a nebulizer filled with 5.0 mL of dosingsolution. Guinea pigs are restrained in the inhalation chamber for 30minutes. During this time, guinea pigs are restricted to an area ofapproximately 110 sq. cm. This space is adequate for the animals to turnfreely, reposition themselves, and allow for grooming. Following 20minutes of acclimation, guinea pigs are exposed to an aerosol generatedfrom a LS Star Nebulizer Set (Model 22F51, PARI Respiratory Equipment,Inc. Midlothian, Va.) driven by house air at a pressure of 22 psi. Uponcompletion of nebulization, guinea pigs are evaluated at 1.5, 6, 12, 24,48, or 72 hrs after treatment.

Guinea pigs are anesthetized one hour before testing with anintramuscular (IM) injection of a mixture of ketamine 43.75 mg/kg,xylazine 3.5 mg/kg, and acepromazine 1.05 mg/kg at an 0.88 mL/kg volume.Animals are placed ventral side up on a heated (37° C.) blanket at a 20degree incline with their head in a downward slope. A 4-ply 2×2 inchgauze pad (Nu-Gauze General-use sponges, Johnson and Johnson, Arlington,Tex.) is inserted in the guinea pig's mouth. Five minutes later, themuscarinic agonist pilocarpine (3.0 mg/kg, SC) is administered and thegauze pad is immediately discarded and replaced by a new pre-weighedgauze pad. Saliva is collected for 10 minutes, at which point the gauzepad is weighed and the difference in weight recorded to determine theamount of accumulated saliva (in mg). The mean amount of salivacollected for animals receiving the vehicle and each dose of testcompound is calculated. The vehicle group mean is considered to be 100%salivation. Results are calculated using result means (n=3 or greater).Confidence intervals (95%) are calculated for each dose at each timepoint using two-way ANOVA. This model is a modified version of theprocedure described in Rechter, “Estimation of anticholinergic drugeffects in mice by antagonism against pilocarpine-induced salivation”Ata Pharmacol Toxicol 24:243-254 (1996).

The mean weight of saliva in vehicle-treated animals, at eachpre-treatment time, is calculated and used to compute % inhibition ofsalivation, at the corresponding pre-treatment time, at each dose. Theinhibition dose-response data are fitted to a four parameter logisticequation using GraphPad Prism, version 3.00 for Windows (GraphPadSoftware, San Diego, Calif.) to estimate anti-sialagogue ID₅₀ (doserequired to inhibit 50% of pilocarpine-evoked salivation). The followingequation is used:Y=Min+(Max−Min)/(1+10^(((log ID50-X)*Hillslope)))where X is the logarithm of dose, Y is the response (% inhibition ofsalivation). Y starts at Min and approaches asymptotically to Max with asigmoidal shape.

The ratio of the anti-sialagogue ID₅₀ to bronchoprotective ID₅₀ is usedto compute the apparent lung-selectivity index of the test compound.Generally, compounds having an apparent lung-selectivity index greaterthan about 5 are preferred. Compounds of the invention are expected tohave an apparent lung-selectivity index greater than 5, when tested inthis or a similar assay.

Assay 5 Methacholine-Induced Depressor Responses in Conscious GuineaPigs

Healthy, adult, male Sprague-Dawley guinea pigs (Harlan, Indianapolis,Ind.), weighing between 200 and 300 g are used in these studies. Underisoflurane anesthesia (to effect), animals are instrumented with commoncarotid artery and jugular vein catheters (PE-50 tubing). The cathetersare exteriorized utilizing a subcutaneous tunnel to the subscapulararea. All surgical incisions are sutured with 4-0 Ethicon Silk and thecatheters locked with heparin (1000 units/mL). Each animal isadministered saline (3 mL, SC) at the end of surgery as well asbuprenorphine (0.05 mg/kg, IM). Animals are allowed to recover on aheating pad before being returned to their holding rooms.

Approximately 18 to 20 hours following surgery, the animals are weighedand the carotid artery catheter on each animal is connected to atransducer for recording arterial pressure. Arterial pressure and heartrate are recorded using a Biopac MP-100 Acquisition System. Animals areallowed to acclimate and stabilize for a period of 20 minutes.

Each animal is challenged with MCh (0.3 mg/kg, IV) administered throughthe jugular venous line and the cardiovascular response is monitored for10 minutes. The animals are then placed into the whole body dosingchamber, which is connected to a nebulizer containing the test compoundor vehicle solution. The solution is nebulized for 10 minutes using agas mixture of breathable air and 5% carbon dioxide with a flow rate of3 liters/minute. The animals are then removed from the whole bodychamber and returned to their respective cages. At 1.5 and 24 hourspost-dosing, the animals are re-challenged with MCh (0.3 mg/kg, IV) andthe hemodynamic response is determined. Thereafter, the animals areeuthanized with sodium pentobarbital (150 mg/kg, IV).

MCh produces a decrease in mean arterial pressure (MAP) and decrease inheart rate (bradycardia). The peak decrease, from baseline, in MAP(depressor responses) is measured for each MCh challenge (before andafter IH dosing). The effects of treatment on the MCh responses areexpressed as % inhibition (mean±SEM) of the control depressor responses.Two-way ANOVA with the appropriate post-hoc test is used to test theeffects of treatment and pre-treatment time. The depressor responses toMCh are expected to be relatively unchanged at 1.5 and 24 hours afterinhalation dosing with vehicle.

The ratio of the anti-depressor ID₅₀ to bronchoprotective ID₅₀ is usedto compute apparent lung-selectivity of the test compound. Generally,compounds having an apparent lung-selectivity index greater than 5 arepreferred. It is expected that the compounds of the invention willexhibit an apparent lung-selectivity index greater than 5, as measuredin this or a similar assay.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statues and regulations, all publications, patents andpatent applications cited herein are hereby incorporated by reference intheir entirety to the same extent as if each document had beenindividually incorporated by reference herein.

1. A compound of the formula:

where: a is 0 or 1; R¹ is halo; b is 0 or 1; R² is halo; A is selectedfrom:

where m is 0 or 1; r is an integer from 2 to 4; and R⁴ is selected fromhydrogen and (1-4C)alkyl; R⁶ is selected from hydrogen and (1-4C)alkyl;X¹ is selected from (1-3C)alkylene and —SO₂; where the alkylene isoptionally substituted with 1 or 2 substituents independently selectedfrom (1 -4C)alkyl and —NR^(Xa)R^(Xb); wherein R^(Xa) and R^(Xb) areindependently selected from hydrogen and (1-4C)alkyl; p is 0 or 1; R⁷represents —OR^(7a), where R^(7a) is (1-4C)alkyl or (3-6C)cycloalkyl;and R⁸ is selected from (1-4C)alkyl, (1-4C)alkyleneNR^(8a)R^(8b), andphenyl, each of R^(8a) and R^(8b) is independently hydrogen or(1-4C)alkyl; or R⁸ is taken together with R⁷ to form a ring having 1 to2 oxygen atoms, where said ring is unsubstituted or substituted by 1 or2 (1-4C)alkyl substituents; wherein each alkyl and alkoxy group in R⁷,R^(7a), R⁸, and R^(8a-8b) is optionally substituted with 1 to 5 fluorosubstituents; or a pharmaceutically acceptable salt or stereoisomerthereof.
 2. The compound of claim 1, wherein a and b each represent 0.3. The compound of claim 1, wherein p is
 0. 4. The compound of claim 1,wherein p is
 1. 5. The compound of claim 1, wherein the —OR⁸ group islocated at the para or meta position.
 6. The compound of claim 1,wherein R⁸ is selected from methyl optionally substituted with 2 to 3fluoro substituents, (1-4C)alkyleneNR^(8a)R^(8b), and phenyl, each ofR^(8a) and R^(8b)is independently methyl or ethyl.
 7. The compound ofclaim 1, wherein R⁸ is taken together with R⁷ to form a ring having 1oxygen atom.
 8. The compound of claim 7, wherein R⁸ is taken togetherwith R⁷ to form —(CH₂)₂—C(CH₃)₂—or —(CH₂₎ ₂—.
 9. The compound of claim1, wherein R⁸ is taken together with R⁷ to form a ring having 2 oxygenatoms.
 10. The compound of claim 9, wherein R⁸ is taken together with R⁷to form —O(CH₂)— or —O(CH₂)₂—.
 11. The compound of claim 1, wherein R⁶is hydrogen or methyl.
 12. The compound of claim 1, wherein A is

m is 0, r is 3, and R⁴ is hydrogen or methyl.
 13. The compound of claim1, having the formula:


14. The compound of claim 1, selected from: biphenyl-2-ylcarbamic acid1-(2-{5-[4-(3-dimethylaminopropoxy)benzylamino]pentylcarbamoyl}ethyl)piperidin-4-ylester; biphenyl-2-ylcarbamic acid1-{2-[5-(4-methoxybenzylamino)pentylcarbamoyl]ethyl}piperidin-4-ylester; biphenyl-2-ylcarbamic acid1-{2-[5-(3-cyclopentyloxy-4-methoxybenzylamino)pentylcarbamoyl]-ethyl}piperidin-4-yl ester; biphenyl-2-ylcarbamic acid1-{2-[5-(3,4-dimethoxybenzylamino)pentylcarbamoyl]ethyl}piperidin-4-ylester; biphenyl-2-ylcarbamic acid1-{2-[5-(4-trifluoromethoxybenzylamino)pentylcarbamoyl]ethyl}piperidin-4-yl ester; biphenyl-2-ylcarbamic acid1-{2-[5-(4-difluoromethoxybenzylamino)pentylcarbamoyl]ethyl}piperidin-4-yl ester; biphenyl-2-ylcarbamic acid1-{2-[5-(4-phenoxybenzylamino)pentylcarbamoyl]-ethyl}piperidin-4-ylester; biphenyl-2-ylcarbamic acid1-(2-{5-[(benzo[1,3]dioxol-5-ylmethyl)amino]pentylcarbamoyl}ethyl)piperidin-4-ylester; biphenyl-2-ylcarbamic acid1-(2-{5-[(2,2-dimethylchroman-6-ylmethyl)amino]pentylcarbamoyl}ethyl)piperidin-4-ylester; biphenyl-2-ylcarbamic acid1-(2-{5-[(2,3-dihydrobenzofuran-5-ylmethyl)amino]pentylcarbamoyl}ethyl)piperidin-4-ylester; biphenyl-2-ylcarbamic acid1-(2-{5-[(2,3-dihydro-benzo[1,4]dioxin-6-ylmethyl)amino]pentylcarbamoyl}ethyl)piperidin-4-yl ester; biphenyl-2-ylcarbamicacid 1-{2-[5-(4-methoxybenzenesulfonylamino)pentylcarbamoyl]ethyl}piperidin-4-yl ester; biphenyl-2-ylcarbamic acid1-(2-{5-[2-(4-methoxyphenyl)ethylamino]pentylcarbamoyl}ethyl)piperidin-4-ylester; and biphenyl-2-ylcarbamic acid1-(2-{5-[2-(3,4-dimethoxyphenyl)ethylamino]pentylcarbamoyl}ethyl)piperidin-4-ylester; or a pharmaceutically acceptable salt thereof.
 15. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of claim 1.